Human Monoclonal Antibodies To O8E

ABSTRACT

The present disclosure provides isolated monoclonal antibodies, particularly human monoclonal antibodies that specifically bind to O8E with high affinity. Nucleic acid molecules encoding the antibodies of this disclosure, expression vectors, host cells and methods for expressing the antibodies of this disclosure are also provided. Immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies of this disclosure are also provided. This disclosure also provides methods for treating cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. Provisional Application Ser.No. 60/748,914, filed on Dec. 8, 2005, and U.S. Provisional ApplicationSer. No. 60/824,593, filed Sep. 5, 2006, both of which are hereinincorporated by reference.

TECHNICAL FIELD

The present disclosure relates generally to the fields of immunology andmolecular biology. More specifically, provided herein are human anti-O8Emonoclonal antibodies, nucleic acids encoding human anti-O8E monoclonalantibodies, methods for preparing human anti-O8E monoclonal antibodiesand methods for the treatment of diseases, such as cancers,characterized by the growth of cells that express O8E.

BACKGROUND

Breast and ovarian cancers are the second and fourth leading causes,respectively, of cancer deaths in females in the United States (AmericanCancer Society (2005) Cancer facts and figures). The American CancerSociety has estimated that, in the United States, approximately 40,000women will die of breast cancer and about 16,000 will die of ovariancancer in 2005. Surface epithelial tumors account for over 80% of allovarian malignancies, which include serous tumors, mucinous tumors,endometrioid tumors and clear cell carcinomas (Seidman et al.“Blaustein's Pathology of the Female Genital Tract” 791-4 (Kurman,editor, 5^(th) ed. New York, Springer-Verlag, 2002). Ovarian cancersfrequently present at an advanced stage where metastatic disease hasspread to regional and distant sites (Pettersson, (1994) Int. Fed. OfGyn. and Obstetrics, Vol. 22; and Heintz et al. (2001) J. Epidermiol.Biostat. 6:107-38). Thus, while the lifetime probability of developingbreast cancer is significantly higher than for ovarian cancer, the 5year survival rate for breast cancer patients is substantially betterthan for those with ovarian cancer.

B7-like molecules belong to the immunoglobulin (Ig) superfamily. Theextracellular portion of B7-like molecules contain single IgV and IgCdomains and share ˜20%-40% amino acid identity. B7-like molecules playcritical roles in the control and fine tuning of antigen-specific immuneresponses. O8E, known also as B7H4, B7x and B7S1, is a member of the B7family and is thought to play a role in both stimulatory and inhibitoryregulation of T cell responses (Carreno et al., (2002) Ann. Rev.Immunol. 20:29-53 and Khoury et al., (2004) Immunity 20:529-538). HumanO8E has been mapped on chromosome 1 and is comprised of six exons andfive introns spanning 66 kb, of which exon 6 is used for alternativesplicing to generate two different transcripts (Choi et al. (2003) J.Immunol. 171:4650-4654).

O8E exerts its physiologic function by binding to a receptor on T cells,which in turn induces cell cycle arrest and inhibits the secretion ofcytokines, the development of cytotoxicity and cytokine production ofCD4⁺ and CD8⁺ T cells (Prasad et al. (2003) Immunity 18:863-873; Sica etal. (2003) Immunity 18:849-861; Wang et al. (2004) Microbes Infect.6:759-66; and Zang et al. (2003) Proc. Natl. Acad. Sci. U.S.A.100:10388-10392). It has been suggested that O8E may be an attenuator ofinflammatory responses and may serve a role in down-regulation ofantigen-specific immune and anti-tumor responses (Zang et al. (2003)Proc. Natl. Acad. Sci. U.S.A. 100:10388-10392; Prasad et al. (2003)Immunity 18:863-873; Sica et al. (2003) Immunity 18:849-861; Choi et al.(2003) J. Immunol. 171:4650-4654; and Carreno et al. (2003) TrendsImmunol. 24:524-7).

O8E mRNA but not protein expression has been detected in a wide range ofnormal somatic tissues, including liver, skeletal muscle, kidney,pancreas and small bowel (Sica et al. (2003) Immunity 18:849-61 and Choiet al. (2003) J. Immunol. 171:4650-4). O8E is inducible upon stimulationof T cells, B cells, monocytes and dendritic cells; however,immunohistochemistry analysis has revealed little expression in severalperipheral tissues with the exception of positive staining in someovarian and lung cancers (Id.). In addition, O8E is consistentlyoverexpressed in primary and metastatic breast cancer, independent oftumor grade or stage, suggesting a critical role for this protein inbreast cancer biology (Tringler et al. (2005) Clinical Cancer Res.11:1842-48). See, also, U.S. Pat. Nos. 6,962,980; 6,699,664; 6,468,546;6,488,931; 6,670,463; and 6,528,253, each of which is incorporated byreference herein in its entirety.

A wide variety of therapeutic modalities are available for the treatmentof advanced breast and ovarian cancers including radiotherapy,conventional chemotherapy with cytotoxic antitumor agents, hormonetherapy (aromatase inhibitors, luteinizing-hormone releasing-hormoneanalogues), bisphosphonates and signal-transduction inhibitors (Smith(2002) Lancet, 360:790-2). Unfortunately, however, many patients eitherrespond poorly or not at all to any of these therapeutic modalities.Thus, there is a need to identify new molecular markers for andtherapeutic agents against breast and ovarian cancers. Accordingly, O8Erepresents a valuable target for the treatment of cancers, includingovarian and breast cancers and a variety of other diseases characterizedby O8E expression.

SUMMARY

The present disclosure provides isolated monoclonal antibodies, inparticular human sequence monoclonal antibodies, that bind to O8E (a/k/aB7H4, B7S1 and B7x) and that exhibit numerous desirable properties.These properties include high affinity binding to human O8E. Alsoprovided are methods for treating a variety of O8E mediated diseasesusing the antibodies and compositions of this disclosure.

In one aspect, this disclosure pertains to an isolated monoclonalantibody or an antigen-binding portion thereof, wherein the antibody:

(a) binds to human O8E with a K_(D) of 1×10⁻⁷ M or less; and

(b) binds to human CHO cells transfected with O8E.

In certain embodiments, the antibody binds to a breast cell carcinomatumor cell line, such as cell line SKBR3 (ATCC Accession No. HTB-30).

Typically the antibody is a human antibody, although in alternativeembodiments the antibody can be a murine antibody, a chimeric antibodyor humanized antibody.

In one embodiment, the antibody binds to human O8E with a K_(D) of5×10⁻⁸ M or less, binds to human O8E with a K_(D) of 2×10⁻⁸ M or less,binds to human O8E with a K_(D) of 1×10⁻⁸ M or less, binds to human O8Ewith a K_(D) of 5×10⁻⁹ M or less, binds to human O8E with a K_(D) of4×10⁻⁹ M or less, binds to human O8E with a K_(D) of 3×10⁻⁹ M or less orbinds to human O8E with a K_(D) of 2×10⁻⁹ M or less.

In another embodiment, the antibody is internalized by SKBR3 breast cellcarcinoma tumor cells after binding to O8E expressed on those cells.

In another embodiment, this disclosure provides an isolated monoclonalantibody or antigen binding portion thereof, wherein the antibodycross-competes for binding to O8E with a reference antibody, wherein thereference antibody:

-   -   (a) binds to human O8E with a K_(D) of 1×10⁻⁷ M or less; and    -   (b) binds to human CHO cells transfected with O8E.        In various embodiments, the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 1; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO: 6;        or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 2; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO: 7.        or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 3; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO: 8 or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 4; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO: 9;        or the reference antibody comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 5; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO: 10.

In one aspect, this disclosure pertains to an isolated monoclonalantibody or an antigen-binding portion thereof, comprising a heavy chainvariable region that is the product of or derived from a human V_(H)4-34 gene (the protein product of which is presented herein as SEQ IDNO: 51), wherein the antibody specifically binds O8E. This disclosurealso provides an isolated monoclonal antibody or an antigen-bindingportion thereof, comprising a heavy chain variable region that is theproduct of or derived from a human V_(H) 3-53 gene (the protein productof which is presented herein as SEQ ID NO: 52), wherein the antibodyspecifically binds O8E. This disclosure also provides an isolatedmonoclonal antibody or an antigen-binding portion thereof, comprising aheavy chain variable region that is the product of or derived from acombination of human V_(H) 3-9/D3-10/JH6b genes (the protein product ofwhich is presented herein as SEQ ID NO: 53), wherein the antibodyspecifically binds O8E.

This disclosure still further provides an isolated monoclonal antibodyor an antigen-binding portion thereof, comprising a light chain variableregion that is the product of or derived from a human V_(K) A27 gene(the protein product of which is presented herein as SEQ ID NO: 54),wherein the antibody specifically binds O8E. This disclosure stillfurther provides an isolated monoclonal antibody or an antigen-bindingportion thereof, comprising a light chain variable region that is theproduct of or derived from a combination of human V_(K) L6/JK1 genes(the protein product of which is presented herein as SEQ ID NO: 55),wherein the antibody specifically binds O8E.

In other aspects, this disclosure provides an isolated monoclonalantibody or an antigen-binding portion thereof, comprising:

-   -   (a) a heavy chain variable region of a human V_(H) 4-34, 3-53 or        3-9 gene; and    -   (b) a light chain variable region of a human V_(K) A27 or V_(K)        L6;    -   wherein the antibody specifically binds to O8E.

In a related embodiment, the antibody comprises a heavy chain variableregion of a human V_(H) 4-34 gene and a light chain variable region of ahuman V_(K) A27 gene. In another related embodiment, the antibodycomprises a heavy chain variable region of a human V_(H) 3-53 gene and alight chain variable region of a human V_(K) A27 gene. In yet anotherrelated embodiment, the antibody comprises a heavy chain variable regionof a human V_(H) 3-9 gene and a light chain variable region of a humanV_(K) L6 gene.

In yet another aspect, the present disclosure provides an isolatedmonoclonal antibody or antigen binding portion thereof, comprising:

-   -   a heavy chain variable region that comprises CDR1, CDR2 and CDR3        sequences; and a light chain variable region that comprises        CDR1, CDR2 and CDR3 sequences, wherein:    -   (a) the heavy chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of amino        acid sequences of SEQ ID NOs: 21, 22, 23, 24 and 25 and        conservative modifications thereof;    -   (b) the light chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of amino        acid sequence of SEQ ID NOs: 36, 37, 38, 39 and 40 and        conservative modifications thereof;    -   (c) the antibody binds to human O8E with a K_(D) of 1×10⁻⁷ M or        less;    -   (d) binds to human CHO cells transfected with O8E.

Typically, the heavy chain variable region CDR2 sequence comprises anamino acid sequence selected from the group consisting of amino acidsequences of SEQ ID NOs: 16, 17, 18, 19 and 20 and conservativemodifications thereof; and the light chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 31, 32, 33, 34 and 35 andconservative modifications thereof. Typically, the heavy chain variableregion CDR1 sequence comprises an amino acid sequence selected from thegroup consisting of amino acid sequences of SEQ ID NOs: 11, 12, 13, 14and 15 and conservative modifications thereof; and the light chainvariable region CDR1 sequence comprises an amino acid sequence selectedfrom the group consisting of amino acid sequences of SEQ ID NOs: 26, 27,28, 29 and 30 and conservative modifications thereof.

A particular combination comprises:

-   -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 11;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 16;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 21;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO: 26;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO: 31;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO: 36.        Another particular combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 12;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 17;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 22;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO: 27;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO: 32;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO: 37.        Another particular combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 13;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 18;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 23;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO: 28;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO: 33;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO: 38.        Another particular combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 14;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 19;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 24;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO: 29;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO: 34;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO: 39.        Another particular combination comprises:    -   (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 15;    -   (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 20;    -   (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 25;    -   (d) a light chain variable region CDR1 comprising SEQ ID NO: 30;    -   (e) a light chain variable region CDR2 comprising SEQ ID NO: 35;        and    -   (f) a light chain variable region CDR3 comprising SEQ ID NO: 40.        Other particular antibodies of this disclosure or antigen        binding portions thereof comprise:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 1; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO: 6.        Another particular combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 2; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO: 7.        Another particular combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 3; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO: 8.        Another particular combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 4; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO: 9.        Another particular combination comprises:    -   (a) a heavy chain variable region comprising the amino acid        sequence of SEQ ID NO: 5; and    -   (b) a light chain variable region comprising the amino acid        sequence of SEQ ID NO: 10.

In another aspect of this disclosure, antibodies or antigen-bindingportions thereof, are provided that compete for binding to O8E with anyof the aforementioned antibodies.

The antibodies of this disclosure can be, for example, full-lengthantibodies, for example of an IgG1, IgG2 or IgG4 isotype. Alternatively,the antibodies can be antibody fragments, such as Fab, Fab′ or Fab′₂fragments or single chain antibodies (e.g., scFv).

This disclosure also provides an immunoconjugate comprising an antibodyof this disclosure or antigen-binding portion thereof, linked to atherapeutic agent, such as a cytotoxin or a radioactive isotope. Thisdisclosure also provides a bispecific molecule comprising an antibody orantigen-binding portion thereof, of this disclosure, linked to a secondfunctional moiety having a different binding specificity than saidantibody or antigen binding portion thereof.

Compositions comprising an antibody or antigen-binding portion thereofor immunoconjugate or bispecific molecule of this disclosure and apharmaceutically acceptable carrier are also provided.

Nucleic acid molecules encoding the antibodies or antigen-bindingportions thereof, of this disclosure are also encompassed by thisdisclosure, as well as expression vectors comprising such nucleic acids,host cells comprising such expression vectors and methods for makinganti-O8E antibodies using such host cells. Moreover, this disclosureprovides a transgenic mouse comprising human immunoglobulin heavy andlight chain transgenes, wherein the mouse expresses an antibody of thisdisclosure, as well as hybridomas prepared from such a mouse, whereinthe hybridoma produces the antibody of this disclosure.

In yet another aspect, this disclosure provides a method of treating orpreventing a disease characterized by growth of tumor cells expressingO8E, comprising administering to a subject an anti-O8E human antibody ofthe present disclosure in an amount effective to treat or prevent thedisease. The disease can be a cancer, e.g., breast cell carcinomacancer.

In yet another aspect, this disclosure provides a method of treating anautoimmune disorder, comprising administering to a subject an anti-O8Ehuman antibody of the present disclosure in an amount effective to treatthe autoimmune disorder.

Other features and advantages of the instant disclosure will be apparentfrom the following detailed description and examples which should not beconstrued as limiting. The contents of all references, Genbank entries,patents and published patent applications cited throughout thisapplication are expressly incorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE IDENTIFIERS

FIG. 1A shows the nucleotide sequence (SEQ ID NO: 41) and amino acidsequence (SEQ ID NO: 1) of the heavy chain variable region of the 1G11human monoclonal antibody. The CDR1 (SEQ ID NO: 11), CDR2 (SEQ ID NO:16) and CDR3 (SEQ ID NO: 21) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 1B shows the nucleotide sequence (SEQ ID NO: 46) and amino acidsequence (SEQ ID NO: 6) of the light chain variable region of the 1G11human monoclonal antibody. The CDR1 (SEQ ID NO: 26), CDR2 (SEQ ID NO:31) and CDR3 (SEQ ID NO: 36) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 2A shows the nucleotide sequence (SEQ ID NO: 42) and amino acidsequence (SEQ ID NO: 2) of the heavy chain variable region of the 2A7human monoclonal antibody. The CDR1 (SEQ ID NO: 12), CDR2 (SEQ ID NO:17) and CDR3 (SEQ ID NO: 22) regions are delineated and the V, D, and Jgermline derivations are indicated.

FIG. 2B shows the nucleotide sequence (SEQ ID NO: 47) and amino acidsequence (SEQ ID NO: 7) of the light chain variable region of the 2A7human monoclonal antibody. The CDR1 (SEQ ID NO: 27), CDR2 (SEQ ID NO:32) and CDR3 (SEQ ID NO: 37) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 3A shows the nucleotide sequence (SEQ ID NO: 43) and amino acidsequence (SEQ ID NO: 3) of the heavy chain variable region of the 2F9human monoclonal antibody. The CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO:18) and CDR3 (SEQ ID NO: 23) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 3B shows the nucleotide sequence (SEQ ID NO: 48) and amino acidsequence (SEQ ID NO: 8) of the light chain variable region of the 2F9human monoclonal antibody. The CDR1 (SEQ ID NO: 28), CDR2 (SEQ ID NO:33) and CDR3 (SEQ ID NO: 38) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 4A shows the nucleotide sequence (SEQ ID NO: 44) and amino acidsequence (SEQ ID NO: 4) of the heavy chain variable region of the 12E1human monoclonal antibody. The CDR1 (SEQ ID NO: 14), CDR2 (SEQ ID NO:19) and CDR3 (SEQ ID NO: 24) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 4B shows the nucleotide sequence (SEQ ID NO: 49) and amino acidsequence (SEQ ID NO: 9) of the light chain variable region of the 12E1human monoclonal antibody. The CDR1 (SEQ ID NO: 29), CDR2 (SEQ ID NO:34) and CDR3 (SEQ ID NO: 39) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 5A shows the nucleotide sequence (SEQ ID NO: 45) and amino acidsequence (SEQ ID NO: 5) of the heavy chain variable region of the 13D12human monoclonal antibody. The CDR1 (SEQ ID NO: 15), CDR2 (SEQ ID NO:20) and CDR3 (SEQ ID NO: 25) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 5B shows the nucleotide sequence (SEQ ID NO: 50) and amino acidsequence (SEQ ID NO: 10) of the light chain variable region of the 13D12human monoclonal antibody. The CDR1 (SEQ ID NO: 30), CDR2 (SEQ ID NO:35) and CDR3 (SEQ ID NO: 40) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 6 shows the alignment of the amino acid sequence of the heavy chainvariable region of 1101 and 13D12 with the human germline V_(H) 4-34amino acid sequence (SEQ ID NO: 51).

FIG. 7 shows the alignment of the amino acid sequence of the heavy chainvariable region of 2A7 and 2F9 with the human germline V_(H) 3-53 aminoacid sequence (SEQ ID NO: 52).

FIG. 8 shows the alignment of the amino acid sequence of the heavy chainvariable region of 12E1 with the combined human germline V_(H)3-91D3-10/JH6b amino acid sequence (SEQ ID NO:53).

FIG. 9 shows the alignment of the amino acid sequence of the light chainvariable region of 1G11, 2A7, 2F9 and 13D12 with the human germlineV_(k) A27 amino acid sequence (SEQ ID NO:54).

FIG. 10 shows the alignment of the amino acid sequence of the lightchain variable region of 12E1 with the combined human germline V_(k)L6/JK1 amino acid sequence (SEQ ID NO:55).

FIGS. 11A and 11B show the results of ELISA experiments demonstratingthat human monoclonal antibodies against human O8E specifically bind toO8E. FIG. 11A shows results from an ELISA plate coated with humananti-O8E antibodies followed by the addition of purified O8E protein anddetection with rabbit anti-O8E antisera. FIG. 11B shows results from anELISA plate coated with anti-mouse Fc followed by monoclonal anti-C9(0.6 μg/ml), then titrated with Penta-O8E protein as indicated andfollowed by human anti-O8E antibodies at 1 μg/ml.

FIG. 12 shows the results of flow cytometry experiments demonstratingthat the anti-08E human monoclonal antibody 2A7 binds to O8E transfectedCHO cells.

FIG. 13 shows the results of flow cytometry experiments demonstratingexpression of O8E in SKBR3 breast carcinoma cells as well as O8Etransfected SKOV3 and HEK cells.

FIG. 14 shows the results of Hum-Zap internalization experimentsdemonstrating that human monoclonal antibodies against human O8E caninternalize into O8E+CHO cells.

FIG. 15 shows the results of Hum-Zap internalization experimentsdemonstrating that human monoclonal antibodies against human O8E caninternalize into O8E⁺ SKBR3 cells.

FIG. 16 shows the results of epitope mapping studies with various humananti-O8E monoclonal antibodies including 1G11, 2A7, 2F9 and 13D12.

FIG. 17 shows the results of antibody dependent cellular cytotoxicity(ADCC) assays demonstrating that human monoclonal anti-O8E antibodieskill human breast cancer cell line SKBR3 in an ADCC dependent manner.

FIG. 18 shows the results of antibody dependent cellular cytotoxicity(ADCC) assays demonstrating that human monoclonal anti-O8E antibodieskill O8E transfected SKOV3 cells in an ADCC dependent manner.

FIG. 19 shows the results of antibody dependent cellular cytotoxicity(ADCC) assays demonstrating that human monoclonal anti-O8E antibodieskill human breast cancer cell line SKBR3 in a concentration and ADCCdependent manner.

FIG. 20 shows the results of in vivo studies on SCID mice showing tumorgrowth inhibition of HEK-B7H4 tumors by anti-O8E antibodies.

DETAILED DESCRIPTION

The present disclosure relates to isolated monoclonal antibodies,particularly human sequence monoclonal antibodies, that bindspecifically to O8E (a/k/a B7H4, B7S1 and B7x) with high affinity. Incertain embodiments, the antibodies of this disclosure are derived fromparticular heavy and light chain germline sequences and/or compriseparticular structural features such as CDR regions comprising particularamino acid sequences. This disclosure provides isolated antibodies,methods of making such antibodies, immunoconjugates and bispecificmolecules comprising such antibodies and pharmaceutical compositionscontaining the antibodies, immunconjugates or bispecific molecules ofthis disclosure. This disclosure also relates to methods of using theantibodies, such as to detect O8E, as well as to treat diseasesassociated with expression of O8E, such as cancer. Accordingly, thisdisclosure also provides methods of using the anti-O8E antibodies ofthis disclosure to treat various cancers, for example, in the treatmentof breast cell carcinomas, metastatic breast cancers, ovarian cellcarcinomas, metastatic ovarian cancers and renal cell carcinomas.

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The terms “O8E,” “B7H4,” “B7x” and “B7 μl” are used hereininterchangeably and include variants, isoforms, homologs, orthologs andparalogs of human O8E. For example, antibodies specific for O8E may, incertain cases, cross-react with O8E from species other than human. Inother embodiments, the antibodies specific for human O8E may becompletely specific for human O8E and may not exhibit species or othertypes of cross-reactivity. The term “human O8E” refers to human sequenceO8E, such as the complete amino acid sequence of human O8E havingGenbank accession number NP_(—)078902 (SEQ ID NO:56). O8E is also knownin the art as, for example, BL-CAM, B3, Leu-14 and Lyb-8. The human O8Esequence may differ from human O8E of SEQ ID NO:56 by having, forexample, conserved mutations or mutations in non-conserved regions andthe CD22 has substantially the same biological function as the human O8Eof SEQ ID NO:56. For example, a biological function of human O8E ishaving an epitope in the extracellular domain of O8E that isspecifically bound by an antibody of the instant disclosure or abiological function of human O8E includes, for example, inhibition ofT-cell proliferation, inhibition of cytokine production, inhibition ofcell cycle production, or binding to T cell receptors.

A particular human O8E sequence will generally be at least 90% identicalin amino acids sequence to human O8E of SEQ ID NO:56 and contains aminoacid residues that identify the amino acid sequence as being human whencompared to O8E amino acid sequences of other species (e.g., murine). Incertain cases, a human 09E may be at least 95%, or even at least 96%,97%, 98%, or 99% identical in amino acid sequence to O8E of SEQ IDNO:56. In certain embodiments, a human O8E sequence will display no morethan 10 amino acid differences from the O8E of SEQ ID NO:56. In certainembodiments, the human O8E may display no more than 5, or even no morethan 4, 3, 2, or 1 amino acid difference from the O8E of SEQ ID NO:56.Percent identity can be determined as described herein.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytesand soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines and complement) that results inselective damage to, destruction of or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. As used herein, the phrase “cell surface receptor”includes, for example, molecules and complexes of molecules capable ofreceiving a signal and the transmission of such a signal across theplasma membrane of a cell. An example of a “cell surface receptor” ofthe present disclosure is the O8E receptor.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e. “antigen-binding portion”) or singlechains thereof. An “antibody” refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds or an antigen binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asV_(H)) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, C_(H1), C_(H2) and C_(H3). Eachlight chain is comprised of a light chain variable region (abbreviatedherein as V_(L)) and a light chain constant region. The light chainconstant region is comprised of one domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

The term “antigen-binding portion” of an antibody (or “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., O8E). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H1)domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fab′fragment, which is essentially an Fab with part of the hinge region (seeFundamental Immunology (Paul ed., 3^(rd) ed. 1993); (iv) a Fd fragmentconsisting of the V_(H) and C_(H1) domains; (v) a Fv fragment consistingof the V_(L) and V_(H) domains of a single arm of an antibody, (vi) adAb fragment (Ward et al., (1989) Nature 341:544-546), which consists ofa V_(H) domain; and (vii) an isolated complementarity determining region(CDR); and (viii) a nanobody, a heavy chain variable region containing asingle variable domain and two constant domains. Furthermore, althoughthe two domains of the Fv fragment, V_(L) and V_(H), are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the V_(L) and V_(H) regions pair to form monovalent molecules(known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art and the fragments are screened forutility in the same manner as are intact antibodies.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds O8E is substantially free of antibodies that specifically bindantigens other than O8E). An isolated antibody that specifically bindsO8E may, however, have cross-reactivity to other antigens, such as O8Emolecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “human antibody” or “human sequence antibody”, as used herein,is intended to include antibodies having variable regions in which boththe framework and CDR regions are derived from human germlineimmunoglobulin sequences. Furthermore, if the antibody contains aconstant region, the constant region also is derived from human germlineimmunoglobulin sequences. The human antibodies may include latermodifications, including natural or synthetic modifications. The humanantibodies of this disclosure may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody,” as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences.

The term “human monoclonal antibody”, which may include the term“sequence” after “human”, refers to antibodies displaying a singlebinding specificity which have variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, the human monoclonal antibodies areproduced by a hybridoma which includes a B cell obtained from atransgenic nonhuman animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by the heavy chain constant region genes.

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.” The term “humanantibody derivatives” refers to any modified form of the human antibody,e.g., a conjugate of the antibody and another agent or antibody.

The term “humanized antibody” is intended to refer to antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. Additional framework region modifications may be made withinthe human framework sequences.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

As used herein, an antibody that “specifically binds to human O8E” isintended to refer to an antibody that binds to human O8E with a K_(D) of1×10⁻⁷ or less, more typically 5×10⁻⁸ M or less, more typically 3×10⁻⁸ Mor less, more typically 1×10⁻⁹ M or less, even more typically 5×10⁻⁹ Mor less.

The term “does not substantially bind” to a protein or cells, as usedherein, means does not bind or does not bind with a high affinity to theprotein or cells, i.e. binds to the protein or cells with a K_(D) of1×10⁻⁶ M or more, more preferably 1×10⁻⁵ M or more, more preferably1×10⁻⁴ M or more, more preferably 1×10⁻³ M or more, even more preferably1×10⁻² M or more.

The term “K_(assoc)” or “K_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “K_(dis)” or “K_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e. K_(d)/K_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A preferred method for determining the K_(D) ofan antibody is by using surface plasmon resonance, typically using abiosensor system such as a Biacore® system.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) of 1×10⁻⁷ M or less, more typically 5×10⁻⁸ Mor less, more typically 1×10⁻⁹ M or less and even more typically 5×10⁻⁹M or less for a target antigen. However, “high affinity” binding canvary for other antibody isotypes. For example, “high affinity” bindingfor an IgM isotype refers to an antibody having a K_(D) of 10⁻⁶ M orless, more typically 10⁻⁷ M or less, even more typically 10⁻⁸ M or less.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows, chickens, amphibians, fish, reptiles, etc.

As used herein, the term “O8E” is used synonymously with the terms“B7H4,” “B7S1,” and “B7x” as these terms variously appear in thescientific literature. The amino acid sequence of O8E (B7H4) is publiclyavailable by reference to GenBank Accession Nos. AAZ17406, AAS13400,AAP37283, CA112739 and CA112737 and by reference to Prasad et al. (2003)Immunity 18:863-873; Sica et al. (2003) Immunity 18:849-861; and U.S.Pat. No. 6,891,030 each of which is incorporated herein by reference inits entirety.

Various aspects of this disclosure are described in further detail inthe following subsections.

Anti-O8E Antibodies

The antibodies of this disclosure are characterized by particularfunctional features or properties of the antibodies. For example, theantibodies bind specifically to human O8E. Typically, an antibody ofthis disclosure binds to O8E with high affinity, for example with aK_(D) of 1×10⁻⁷ M or less. The anti-O8E antibodies of this disclosuretypically exhibit one or more of the following characteristics:

(a) binds to human O8E with a K_(D) of 1×10⁻⁷ M or less;

(b) binds to human CHO cells transfected with O8E.

Typically, the antibody binds to human O8E with a K_(D) of 5×10⁻⁸ M orless, bind to human O8E with a K_(D) of 2×10⁻⁸ M or less, binds to humanO8E with a K_(D) of 5×10⁻⁹ M or less, binds to human O8E with a K_(D) of4×10⁻⁹ M or less, binds to human O8E with a K_(D) of 3×10⁻⁹ M or less,binds to human O8E with a K_(D) of 2×10⁻⁹ M or less or binds to humanO8E with a K_(D) of 1×10⁻⁹ M or less.

Standard assays to evaluate the binding ability of the antibodies towardO8E are known in the art, including for example, ELISAs, Western blots,RIAs and flow cytometry analysis. Suitable assays are described indetail in the Examples. The binding kinetics (e.g., binding affinity) ofthe antibodies also can be assessed by standard assays known in the art,such as by ELISA, Scatchard and Biacore® system analysis. As anotherexample, the antibodies of the present disclosure may bind to a breastcarcinoma tumor cell line, for example, the SKBR3 cell line.

Monoclonal Antibodies 1G11, 2A7, 2F9, 12E1 and 13D12

Exemplified antibodies of this disclosure include the human monoclonalantibodies 1G11, 2A7, 2F9, 12E1 and 13D12 isolated and structurallycharacterized as described in Examples 1 and 2. The V_(H) amino acidsequences of 1G11, 2A7, 2F9, 12E1 and 13D12 are shown in SEQ ID NOs: 1,2, 3, 4 and, 5 respectively. The V_(L) amino acid sequences of 1G11,2A7, 2F9, 12E1 and 13D12 are shown in SEQ ID NOs: 6, 7, 8, 9 and 10,respectively.

Given that each of these antibodies can bind to O8E, the V_(H) and V_(L)sequences can be “mixed and matched” to create other anti-O8E bindingmolecules of this disclosure. O8E binding of such “mixed and matched”antibodies can be tested using the binding assays described above and inthe Examples (e.g., FACS or ELISAs). Typically, when V_(H) and V_(L)chains are mixed and matched, a V_(H) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(H)sequence. Likewise, typically a V_(L) sequence from a particularV_(H)/V_(L) pairing is replaced with a structurally similar V_(L)sequence.

Accordingly, in one aspect, this disclosure provides an isolatedmonoclonal antibody or antigen binding portion thereof comprising:

(a) a heavy chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1, 2, 3, 4 and 5; and

(b) a light chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 6, 7, 8, 9 and 10;wherein the antibody specifically binds to O8E, typically human O8E.

Preferred heavy and light chain combinations include:

(a) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 1; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 6; or

(b) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 2; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 7; or

(c) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 3; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 8;

(d) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 4; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 9; or

(e) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 5; and (b) a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 10.

In another aspect, this disclosure provides antibodies that comprise theheavy chain and light chain CDR1s, CDR2s and CDR3s of G101, 2A7, 2F9,12E1 and 13D12 or combinations thereof. The amino acid sequences of theV_(H) CDR1s of 10G1, 2A7, 2F9, 12E1 and 13D12 are shown in SEQ ID NOs:11, 12, 13, 14 and 15, respectively. The amino acid sequences of theV_(H) CDR2s of 1G11, 2A7, 2F9, 12E1 and 13D12 are shown in SEQ ID NOs:16, 17, 18, 19 and 20, respectively. The amino acid sequences of theV_(H) CDR3s of 1G11, 2A7, 2F9, 12E1 and 13D12 are shown in SEQ ID NOs:21, 22, 23, 24 and 25, respectively. The amino acid sequences of theV_(k) CDR1s of 1G11, 2A7, 2F9, 12E1 and 13D12 are shown in SEQ ID NOs:26, 27, 28, 29 and 30, respectively. The amino acid sequences of theV_(k) CDR2s of 1G11, 2A7, 2F9, 12E1 and 13D12 are shown in SEQ ID NOs:31, 32, 33, 34 and 35, respectively. The amino acid sequences of theV_(k) CDR3s of 1G11, 2A7, 2F9, 12E1 and 13D12 are shown in SEQ ID NOs:36, 37, 38, 39 and 40, respectively. The CDR regions are delineatedusing the Kabat system (Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242).

Each of the above referenced amino acid and nucleotide sequences of thehuman antibodies designated herein as 1G11, 2A7, 2F9, 12E1 and 13D12 arepresented in the following Table 1 and Sequence Listing,

TABLE 1 Sequences of Heavy and Light Chain Variable and Constant Regionsand Corresponding CDRs of Human Antibodies 1G11, 2A7, 2F9, 12E1 and13D12 Sequence Identifier Description Sequence SEQ ID NO: 1 Amino acidsequence of QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYFWTWIRQP the heavy chainvariable PGKGLEWIGEINHSGTTNYNPSLKSRVTISADTSKNQFSR region of the 1G11human LSSVTAADTAVYYCARLSSWSNWAFEYWGQGTLVTVSS monoclonal antibody SEQ IDNO: 2 Amino acid sequence of EVQLVESGGGLIQPGGSLRLSCAASGFTVSSNYMNWVRQAthe heavy chain variable PGKGLEWVSVIYGSGRTYYADSVKGRVTISRDNSKNTLYL regionof the 2A7 human QMNSLRAEDTAVYYCARDTYAMDVWGQGTTVTVSS monoclonal antibodySEQ ID NO: 3 Amino acid sequence ofEVQLVESGGGLIQPGGSLRLSCAASGFIVSRNYMNWVRQA the heavy chain variablePGKGLEWVSVIYGSGRTDCADSVKGRFTISRDNSKNTLYL region of the 2F9 humanQMNSLRAEDTAVYYCARDGDYGMDVWGQGTTVTVSS monoclonal antibody SEQ ID NO: 4Amino acid sequence of EVQLVESGGGLVQPGRSLRLSCVASGFTFDDYAMHWVRQA theheavy chain variable PGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLY region ofthe 12E1 human LQMNSLRAEDTALYYCTKALYGSGSSDFYYYGMDVWGQGT monoclonalantibody TVAVSS SEQ ID NO: 5 Amino acid sequence ofQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQP the heavy chain variablePGKGLEWIGKINHSGSTNYNPSLKSRVTISVDTSKNQFSL region of the 13D12KLNSVTAADTAVYYCARELRYFENYYYGMDVWGQGTTVTV human monoclonal SS antibodySEQ ID NO: 6 Amino acid sequence ofEIVLTQFPGTLSLSPGERATLSCRASQSVSSTYLAWYQQK the light chain variablePGQAPRVLIYGASRRATGIPDRFSGSGSGTDFTLTISRLE region of the 1G11 humanPEDFAVYYCQQYGSSPLTFGGGTKVEIK monoclonal antibody SEQ ID NO: 7 Amino acidsequence of EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQK the light chainvariable PGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLE region of the 2A7human PEDFAVYYCQQYGSSPMYTFGQGTKLEIK monoclonal antibody SEQ ID NO: 8Amino acid sequence of EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQK thelight chain variable PGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLE region ofthe 2F9 human PEDFAVYYCQQYGSSPLYTFGQGTKLEIK monoclonal antibody SEQ IDNO: 9 Amino acid sequence of EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPthe light chain variable GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP regionof the 12E1 human EDFAVYYCQQRRTFGQGTKVEIK monoclonal antibody SEQ ID NO:10 Amino acid sequence of EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQK thelight chain variable PGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLE region ofthe 13D12 PEDFAVYYCQQYGSSPRTFGQGTKVEIK human monoclonal antibody SEQ IDNO: 11 Amino acid sequence of DYFWT the heavy chain variable region CDR1of the 1G11 human monoclonal antibody SEQ ID NO: 12 Amino acid sequenceSNYMNW the heavy chain variable region CDRl of the 2A7 human monoclonalantibody SEQ ID NO: 13 Amino acid sequence of RNYMN the heavy chainvariable region CDR1 of the 2F9 human monoclonal antibody SEQ ID NO: 14Amino acid sequence of DYAMH the heavy chain variable region CDR1 of the12E1 human monoclonal antibody SEQ ID NO: 15 Amino acid sequence ofGYYWS the heavy chain variable region CDRl of the 13D12 human monoclonalantibody SEQ ID NO: 16 Amino acid sequence of EINHSGTTNYNPSLKS the heavychain variable region CDR2 of the 1G11 human monoclonal antibody SEQ IDNO: 17 Amino acid sequence of VIYGSGRTYYADSVKG the heavy chain variableregion CDR2 of the 2A7 human monoclonal antibody SEQ ID NO: 18 Aminoacid sequence of VIYGSGRTDCADSVKG the heavy chain variable region CDR2of the 2F9 human monoclonal antibody SEQ ID NO: 19 Amino acid sequenceof GISWNSGSIGYADSVKG the heavy chain variable region CDR2 of the 12E1human monoclonal antibody SEQ ID NO: 20 Amino acid sequence ofKINHSGSTNYNPSLKS the heavy chain variable region CDR2 of the 13D12 humanmonoclonal antibody SEQ ID NO: 21 Amino acid sequence of LSSWSNWAFEY theheavy chain variable region CDR3 of the 1G11 human monoclonal antibodySEQ ID NO: 22 Amino acid sequence of DTYANDV the heavy chain variableregion CDR3 of the 2A7 human monoclonal antibody SEQ ID NO: 23 Aminoacid sequence of DGDYGMDV the heavy chain variable region CDR3 of the2F9 human monoclonal antibody SEQ ID NO: 24 Amino acid sequence ofLYGSGSSDFYYYGMDV the heavy chain variable region CDR3 of the 12E1 humanmonoclonal antibody SEQ ID NO: 25 Amino acid sequence of ELRYFENYYYGMDVthe heavy chain variable region CDR3 of the 13D12 human monoclonalantibody SEQ ID NO: 26 Amino acid sequence of RASQSVSSTYLA the lightchain variable region CDR1 of the 1G11 human monoclonal antibody SEQ IDNO: 27 Amino acid sequence of RASQSVSSSYLA the light chain variableregion CDR1 of the 2A7 human monoclonal antibody SEQ ID NO: 28 Aminoacid sequence of RASQSVSSSYLA the light chain variable region CDR1 ofthe 2F9 human monoclonal antibody SEQ ID NO: 29 Amino acid sequence ofRASQSVSSYLA the light chain variable region CDR1 of the 12E1 humanmonoclonal antibody SEQ ID NO: 30 Amino acid sequence of RASQSVSSSYLAthe light chain variable region CDR1 of the 13D12 human monoclonalantibody SEQ ID NO: 31 Amino acid sequence of GASRRAT the light chainvariable region CDR2 of the 1G11 human monoclonal antibody SEQ ID NO: 32Amino acid sequence of GASSRAT the light chain variable region CDR2 ofthe 2A7 human monoclonal antibody SEQ ID NO: 33 Amino acid sequence ofGASSRAT the light chain variable region CDR2 of the 2F9 human monoclonalantibody SEQ ID NO: 34 Amino acid sequence of DASNRAT the light chainvariable region CDR2 of the 12E1 human monoclonal antibody SEQ ID NO: 35Amino acid sequence of GASSRAT the light chain variable region CDR2 ofthe 13D12 human monoclonal antibody SEQ ID NO: 36 Amino acid sequence ofQQYGSSPLT the light chain variable region CDR3 of the 1G11 humanmonoclonal antibody SEQ ID NO: 37 Amino acid sequence of QQYGSSPMYT thelight chain variable region CDR3 of the 2A7 human monoclonal antibodySEQ ID NO: 38 Amino acid sequence of QQYGSSPLYT the light chain variableregion CDR3 of the 2F9 human mono clonal antibody SEQ ID NO: 39 Aminoacid sequence of QQRRT the light chain variable region CDR3 of the 12E1human monoclonal antibody SEQ ID NO: 40 Amino acid sequence of QQYGSSPRTthe light chain variable region CDR3 of the 13D12 human monoclonalantibody SEQ ID NO: 41 Nucleotide sequence ofthe heavy chainvariableregion of the 1G11 humanmonoclonal antibody

SEQ ID NO: 42 Nucleotide sequence ofthe heavy chain variableregion ofthe 2A7 humanmonoclonal antibody

SEQ ID NO: 43 Nucleotide sequence ofthe heavy chain variableregion ofthe 2F9 humanmonoclonal antibody

SEQ ID NO: 44 Nucleotide sequence ofGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGC the heavy chain variableCTGGCAGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATT region of the 12E1 humanCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCT monoclonal antibodyCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTACAAAAGCCCTCTATGGTTCGGGGAGTTCTGACTTCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACC ACGGTCGCCGTCTCCTCA SEQ ID NO:45 Nucleotide sequence of CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGC theheavy chain variable CTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGG region ofthe 13D12 GTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCC human monoclonalCCAGGGAAGGGGCTGGAGTGGATTGGGAAAATCAATCATA antibodyGCGGAAGTACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAACTAAACTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGAATTACGATATTTTGAAAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTC TCCTCA SEQ ID NO: 46 Nucleotidesequence ofthe light chain variableregion of the 1G11 humanmonoclonalantibody

SEQ ID NO: 47 Nucleotide sequence ofGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGT the light chain variableCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCA region of the 2A7 humanGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAA monoclonal antibodyCCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCCATGTACACTTTTGGCCAGGGGACCAAGCTGGA GATCAAA SEQ ID NO: 48Nucleotide sequence ofthe light chain variableregion of the 2F9humanmonoclonal antibody

SEQ ID NO: 49 Nucleotide sequence ofthe light chain variableregion ofthe 12E1 humanmonoclonal antibody

SEQ ID NO: 50 Nucleotide sequence ofGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGT the light chain variableCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCA region of the 13D12GAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAA human monoclonalCCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCA antibodyGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAAT CAAA SEQ ID NO: 51 Amino acidsequence of QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQP the human germlineV_(H) 4- PGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSL 34 KLSSVTAADTAVYYCARSEQ ID NO: 52 Amino acid sequence ofEVQLVESGGGLIQPGGSLRLSCAASGFTVSSNYMSWVRQA the human germline V_(H) 3-PGKGLEWVSVIYSGGSTYYADSVKGRFTISRDNSKNTLYL 53 QMNSLRAEDTAVYYCAR SEQ ID NO:53 Amino acid sequence of EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQA thehuman germline V_(H) 3- PGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLY9/D3-10/JH6b LQMNSLRAEDTALYYCAKDYGSGSYYYYYGMDVWGQGTTV TVSS SEQ ID NO: 54Amino acid sequence of EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQK thehuman germline V_(k) PGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLE A27PEDFAVYYCQQYGSSP SEQ ID NO: 55 Amino acid sequence ofEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP the human germline V_(k)GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP L6/JK1EDFAVYYCQQRSNWTFGQGTKVEIK

Given that each of the human antibodies designated 1G11, 2A7, 2F9, 12E1and 13D12 can bind to O8E and that antigen-binding specificity isprovided primarily by the CDR1, CDR2 and CDR3 regions, the V_(H) CDR1,CDR2 and CDR3 sequences and V_(k) CDR1, CDR2 and CDR3 sequences can be“mixed and matched” (i.e. CDRs from different antibodies can be mixedand matched, although each antibody must contain a V_(H) CDR1, CDR2 andCDR3 and a V_(k) CDR1, CDR2 and CDR3) to create other anti-O8E bindingmolecules of this disclosure. O8E binding of such “mixed and matched”antibodies can be tested using the binding assays described above and inthe Examples (e.g., FACS, ELISAs, Biacore® system analysis). Typically,when V_(H) CDR sequences are mixed and matched, the CDR1, CDR2 and/orCDR3 sequence from a particular V_(H) sequence is replaced with astructurally similar CDR sequence(s). Likewise, when V_(k) CDR sequencesare mixed and matched, the CDR1, CDR2 and/or CDR3 sequence from aparticular V_(k) sequence typically is replaced with a structurallysimilar CDR sequence(s). It will be readily apparent to the ordinarilyskilled artisan that novel V_(H) and V_(L) sequences can be created bysubstituting one or more V_(H) and/or V_(L) CDR region sequences withstructurally similar sequences from the CDR sequences disclosed hereinfor monoclonal antibodies 1G11, 2A7, 2F9, 12E1 and 13D12.

Accordingly, in another aspect, this disclosure provides an isolatedmonoclonal antibody or antigen binding portion thereof comprising:

(a) a heavy chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 11, 12, 13, 14 and 15;

(b) a heavy chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 16, 17, 18, 19 and 20;

(c) a heavy chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 21, 22, 23, 24 and 25;

(d) a light chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 26, 27, 28, 29 and 30;

(e) a light chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 31, 32, 33, 34 and 35;and

(f) a light chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 36, 37, 38, 39 and 40;wherein the antibody specifically binds O8E, typically human O8E.

In a preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 11;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 16;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 21;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 26;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 31; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 36.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 12;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 17;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 22;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 27;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 32; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 37.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 13;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 18;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 23;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 28;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 33; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 38.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 14;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 19;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 24;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 29;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 34; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 39.

In another preferred embodiment, the antibody comprises:

(a) a heavy chain variable region CDR1 comprising SEQ ID NO: 15;

(b) a heavy chain variable region CDR2 comprising SEQ ID NO: 20;

(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 25;

(d) a light chain variable region CDR1 comprising SEQ ID NO: 30;

(e) a light chain variable region CDR2 comprising SEQ ID NO: 35; and

(f) a light chain variable region CDR3 comprising SEQ ID NO: 40.

It is well known in the art that the CDR3 domain, independently from theCDR1 and/or CDR2 domain(s), alone can determine the binding specificityof an antibody for a cognate antigen and that multiple antibodies canpredictably be generated having the same binding specificity based on acommon CDR3 sequence. See, for example, Klimka et al., British J. ofCancer 83(2):252-260 (2000) (describing the production of a humanizedanti-CD30 antibody using only the heavy chain variable domain CDR3 ofmurine anti-CD30 antibody Ki-4); Beiboer et al., J. Mol. Biol.296:833-849 (2000) (describing recombinant epithelial glycoprotein-2(EGP-2) antibodies using only the heavy chain CDR3 sequence of theparental murine MOC-31 anti-EGP-2 antibody); Rader et al., Proc. Natl.Acad. Sci. U.S.A. 95:8910-8915 (1998) (describing a panel of humanizedanti-integrin α_(v)β₃ antibodies using a heavy and light chain variableCDR3 domain of a murine anti-integrin α_(v)β₃ antibody LM609 whereineach member antibody comprises a distinct sequence outside the CDR3domain and capable of binding the same epitope as the parent muringantibody with affinities as high or higher than the parent murineantibody); Barbas et al., J. Am. Chem. Soc. 116:2161-2162 (1994)(disclosing that the CDR3 domain provides the most significantcontribution to antigen binding); Barbas et al., Proc. Natl. Acad. Sci.USA. 92:2529-2533 (1995) (describing the grafting of heavy chain CDR3sequences of three Fabs (SI-1, SI-40 and SI-32) against human placentalDNA onto the heavy chain of an anti-tetanus toxoid Fab thereby replacingthe existing heavy chain CDR3 and demonstrating that the CDR3 domainalone conferred binding specificity); and Ditzel et al., J. Immunol.157:739-749 (1996) (describing grafting studies wherein transfer of onlythe heavy chain CDR3 of a parent polyspecific Fab LNA3 to a heavy chainof a monospecific IgG tetanus toxoid-binding Fab p313 antibody wassufficient to retain binding specificity of the parent Fab). Each ofthese references is hereby incorporated by reference in its entirety.

Accordingly, within certain aspects, the present disclosure providesmonoclonal antibodies comprising one or more heavy and/or light chainCDR3 domain from a non-human antibody, such as a mouse or rat antibody,wherein the monoclonal antibody is capable of specifically binding toO8E. Within some embodiments, such inventive antibodies comprising oneor more heavy and/or light chain CDR3 domain from a non-human antibody(a) are capable of competing for binding with; (b) retain the functionalcharacteristics; (c) bind to the same epitope; and/or (d) have a similarbinding affinity as the corresponding parental non-human antibody.

Within other aspects, the present disclosure provides monoclonalantibodies comprising one or more heavy and/or light chain CDR3 domainfrom a first human antibody, such as, for example, a human antibodyobtained from a non-human animal, wherein the first human antibody iscapable of specifically binding to O8E and wherein the CDR3 domain fromthe first human antibody replaces a CDR3 domain in a human antibody thatis lacking binding specificity for O8E to generate a second humanantibody that is capable of specifically binding to O8E. Within someembodiments, such inventive antibodies comprising one or more heavyand/or light chain CDR3 domain from the first human antibody (a) arecapable of competing for binding with; (b) retain the functionalcharacteristics; (c) bind to the same epitope; and/or (d) have a similarbinding affinity as the corresponding parental first human antibody.

Antibodies Having Particular Germline Sequences

In certain embodiments, an antibody of this disclosure comprises a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene.

For example, in a preferred embodiment, this disclosure provides anisolated monoclonal antibody or an antigen-binding portion thereof,comprising a heavy chain variable region that is the product of orderived from a human V_(H) 4-34 gene, wherein the antibody specificallybinds O8E. In another preferred embodiment, this disclosure provides anisolated monoclonal antibody or an antigen-binding portion thereof,comprising a heavy chain variable region that is the product of orderived from a human V_(H) 3-53 gene, wherein the antibody specificallybinds O8E. In another preferred embodiment, this disclosure provides anisolated monoclonal antibody or an antigen-binding portion thereof,comprising a heavy chain variable region that is the product of orderived from a combined human V_(H) 3-9/D3-10/JH6b gene, wherein theantibody specifically binds O8E.

In another preferred embodiment, this disclosure provides an isolatedmonoclonal antibody or an antigen-binding portion thereof, comprising alight chain variable region that is the product of or derived from ahuman V_(K) A27 gene, wherein the antibody specifically binds O8E. Inanother preferred embodiment, this disclosure provides an isolatedmonoclonal antibody or an antigen-binding portion thereof, comprising alight chain variable region that is the product of or derived from acombined human V_(K) L6/JK1 gene, wherein the antibody specificallybinds O8E.

In yet another preferred embodiment, this disclosure provides anisolated monoclonal antibody or antigen-binding portion thereof, whereinthe antibody:

(a) comprises a heavy chain variable region that is the product of orderived from a human V_(H) 4-34 gene, a human V_(H) 3-53 gene or acombined human V_(H) 3-9/D3-10/JH6b gene (which genes encode the aminoacid sequences set forth in SEQ ID NOs: 51, 52 and 53, respectively);

(b) comprises a light chain variable region that is the product of orderived from a human V_(K) A27 gene or a combined human V_(K) L6/JK1gene (which genes encode the amino acid sequences set forth in SEQ IDNOs: 54 and 55, respectively); and

(c) the antibody specifically binds to O8E, typically human O8E.

Examples of antibodies having V_(H) and V_(K) of V_(H) 4-34 and V_(K)A27, respectively, are 1G11 and 13D12. Examples of antibodies havingV_(H) and V_(K) of V_(H) 3-53 and V_(K) A27, respectively, are 2A7 and2F9. An example of an antibody having V_(H) and V_(K) of V_(H) 3-9/D3-10/JH6b and V_(K) L6/JK1, respectively, is 12E1.

As used herein, a human antibody comprises heavy or light chain variableregions that is “the product of” or “derived from” a particular germlinesequence if the variable regions of the antibody are obtained from asystem that uses human germline immunoglobulin genes. Such systemsinclude immunizing a transgenic mouse carrying human immunoglobulingenes with the antigen of interest or screening a human immunoglobulingene library displayed on phage with the antigen of interest. A humanantibody that is “the product of” or “derived from” a human germlineimmunoglobulin sequence can be identified as such by comparing the aminoacid sequence of the human antibody to the amino acid sequences of humangermline immunoglobulins and selecting the human germline immunoglobulinsequence that is closest in sequence (i.e. greatest % identity) to thesequence of the human antibody. A human antibody that is “the productof” or “derived from” a particular human germline immunoglobulinsequence may contain amino acid differences as compared to the germlinesequence, due to, for example, naturally-occurring somatic mutations orintentional introduction of site-directed mutation. However, a selectedhuman antibody typically is at least 90% identical in amino acidssequence to an amino acid sequence encoded by a human germlineimmunoglobulin gene and contains amino acid residues that identify thehuman antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a human antibody may be at least95% or even at least 96%, 97%, 98% or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5 or even no more than 4, 3, 2 or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

Homologous Antibodies

In yet another embodiment, an antibody of this disclosure comprisesheavy and light chain variable regions comprising amino acid sequencesthat are homologous to the amino acid sequences of the preferredantibodies described herein and wherein the antibodies retain thedesired functional properties of the anti-O8E antibodies of thisdisclosure.

For example, this disclosure provides an isolated monoclonal antibody orantigen binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein:

(a) the heavy chain variable region comprises an amino acid sequencethat is at least 80% homologous to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1, 2, 3, 4; and 5

(b) the light chain variable region comprises an amino acid sequencethat is at least 80% homologous to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 6, 7, 8; 9and 10;

(c) the antibody binds to human O8E with a K_(D) of 1×10⁻⁷ M or less;and

(d) the antibody binds to human CHO cells transfected with O8E.

In various embodiments, the antibody can be, for example, a humanantibody, a humanized antibody or a chimeric antibody.

In other embodiments, the V_(H) and/or V_(L) amino acid sequences may be85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences setforth above. An antibody having V_(H) and V_(L) regions having high(i.e. 80% or greater) homology to the V_(H) and V_(L) regions of thesequences set forth above, can be obtained by mutagenesis (e.g.,site-directed or PCR-mediated mutagenesis) of nucleic acid moleculesencoding SEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50, followedby testing of the encoded altered antibody for retained function (i.e.,the functions set forth in (c) and (d) above), using the functionalassays described herein.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e. % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weightof 1, 2, 3, 4, 5 or 6.

Additionally or alternatively, the protein sequences of the presentdisclosure can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.Such searches can be performed using the XBLAST program (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the antibody molecules of thisdisclosure. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of this disclosure comprises a heavychain variable region comprising CDR1, CDR2 and CDR3 sequences and alight chain variable region comprising CDR1, CDR2 and CDR3 sequences,wherein one or more of these CDR sequences comprise specified amino acidsequences based on the preferred antibodies described herein (e.g.,1G11, 2A7, 2F9, 12E1 or 13D12) or conservative modifications thereof andwherein the antibodies retain the desired functional properties of theanti-O8E antibodies of this disclosure. Accordingly, this disclosureprovides an isolated monoclonal antibody or antigen binding portionthereof, comprising a heavy chain variable region comprising CDR1, CDR2and CDR3 sequences and a light chain variable region comprising CDR1,CDR2 and CDR3 sequences, wherein:

-   -   (a) the heavy chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of amino        acid sequences of SEQ ID NOs: 21, 22, 23, 24 and 25 and        conservative modifications thereof,    -   (b) the light chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of amino        acid sequence of SEQ ID NOs: 36, 37, 38, 39 and 40 and        conservative modifications thereof;    -   (c) the antibody binds to human O8E with a K_(D) of 1×10⁻⁷ M or        less; and    -   (d) the antibody binds to human CHO cells transfected with O8E.

In a preferred embodiment, the heavy chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 16, 17, 18, 19 and 20 andconservative modifications thereof; and the light chain variable regionCDR2 sequence comprises an amino acid sequence selected from the groupconsisting of amino acid sequences of SEQ ID NOs: 31, 32, 33, 34 and 35and conservative modifications thereof. In another preferred embodiment,the heavy chain variable region CDR1 sequence comprises an amino acidsequence selected from the group consisting of amino acid sequences ofSEQ ID NOs: 11, 12, 13, 14 and 15 and conservative modificationsthereof; and the light chain variable region CDR1 sequence comprises anamino acid sequence selected from the group consisting of amino acidsequences of SEQ ID NOs: 26, 27, 28, 29 and 30 and conservativemodifications thereof.

In various embodiments, the antibody can be, for example, humanantibodies, humanized antibodies or chimeric antibodies.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of this disclosure by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody ofthis disclosure can be replaced with other amino acid residues from thesame side chain family and the altered antibody can be tested forretained function.

Antibodies that Bind to the Same Epitope as Anti-O8E Antibodies of thisDisclosure

In another embodiment, this disclosure provides antibodies that bind tothe same epitope on human O8E as any of the O8E monoclonal antibodies ofthis disclosure (i.e. antibodies that have the ability to cross-competefor binding to O8E with any of the monoclonal antibodies of thisdisclosure). In preferred embodiments, the reference antibody forcross-competition studies can be the monoclonal antibody 1G11 (havingV_(H) and V_(L) sequences as shown in SEQ ID NOs: 1 and 6, respectively)or the monoclonal antibody 2A7 (having V_(H) and V_(L) sequences asshown in SEQ ID NOs: 2 and 7, respectively) or the monoclonal antibody2F9 (having V_(H) and V_(L) sequences as shown in SEQ ID NOs: 3 and 8,respectively) or the monoclonal antibody 12E1 (having V_(H) and V_(L)sequences as shown in SEQ ID NOs: 4 and 9, respectively) or themonoclonal antibody 13D12 (having V_(H) and V_(L) sequences as shown inSEQ ID NOs: 5 and 10, respectively). Such cross-competing antibodies canbe identified based on their ability to cross-compete with 1G11, 2A7,2F9, 12E1 or 13D12 in standard O8E binding assays. For example, BIAcore®system analysis, ELISA assays or flow cytometry may be used todemonstrate cross-competition with the antibodies of the currentdisclosure. The ability of a test antibody to inhibit the binding of,for example, 111, 2A7, 2F9, 12 μl or 13D12 to human O8E demonstratesthat the test antibody can compete with 1G11, 2A7, 2F9, 12E1 or 13D12for binding to human O8E and thus binds to the same epitope on human O8Eas 1G11, 2A7, 2F9, 12E1 or 13D12. In a preferred embodiment, theantibody that binds to the same epitope on human O8E as 1G11, 2A7, 2F9,12E1 or 13D12 is a human monoclonal antibody. Such human monoclonalantibodies can be prepared and isolated as described in the Examples.

Engineered and Modified Antibodies

An antibody of this disclosure further can be prepared using an antibodyhaving one or more of the V_(H) and/or V_(L) sequences disclosed hereinas starting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i.e. V_(H) and/or V_(L)), for example withinone or more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. etal. (1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.)

Accordingly, another embodiment of this disclosure pertains to anisolated monoclonal antibody or antigen binding portion thereof,comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3sequences comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 11, 12, 13, 14 and 15; SEQ ID NOs: 16, 17, 18,19 and 20; and SEQ ID NOs: 21, 22, 23, 24 and 25; respectively and alight chain variable region comprising CDR1, CDR2 and CDR3 sequencescomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 26, 27, 28, 29 and 30; SEQ ID NOs: 31, 32, 33, 34 and 35;and SEQ ID NOs: 36, 37, 38, 39 and 40; respectively. Thus, suchantibodies contain the V_(H) and V_(L) CDR sequences of monoclonalantibodies 1G11, 2A7, 2F9, 12E1 or 13D12 yet may contain differentframework sequences from these antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.(1992) “The Repertoire of Human Germline V_(H) Sequences Reveals aboutFifty Groups of V_(H) Segments with Different Hypervariable Loops” J.Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) “A Directory ofHuman Germ-line V_(H) Segments Reveals a Strong Bias in their Usage”Eur. J. Immunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference. As another example, the germline DNAsequences for human heavy and light chain variable region genes can befound in the Genbank database. For example, the following heavy chaingermline sequences found in the HCo7 HuMAb mouse are available in theaccompanying Genbank accession numbers: 1-69 (NG_(—)0010109,NT_(—)024637 and BC070333), 3-33 (NG_(—)0010109 and NT_(—)024637) and3-7 (NG_(—)0010109 and NT_(—)024637). As another example, the followingheavy chain germline sequences found in the HCo12 HuMAb mouse areavailable in the accompanying Genbank accession numbers: 1-69(NG_(—)0010109, NT_(—)024637 and BC070333), 5-51 (NG_(—)0010109 andNT_(—)024637), 4-34 (NG_(—)0010109 and NT_(—)024637), 3-30.3 (CAJ556644)and 3-23 (AJ406678).

Antibody protein sequences are compared against a compiled proteinsequence database using one of the sequence similarity searching methodscalled the Gapped BLAST (Altschul et al. (1997) Nucleic Acids Research25:3389-3402), which is well known to those skilled in the art. BLAST isa heuristic algorithm in that a statistically significant alignmentbetween the antibody sequence and the database sequence is likely tocontain high-scoring segment pairs (HSP) of aligned words. Segment pairswhose scores cannot be improved by extension or trimming is called ahit. Briefly, the nucleotide sequences of VBASE origin(http://vbase.mrc-cpe.cam.ac.uk/vbase1/list2.php) are translated and theregion between and including FR1 through FR3 framework region isretained. The database sequences have an average length of 98 residues.Duplicate sequences which are exact matches over the entire length ofthe protein are removed. A BLAST search for proteins using the programblastp with default, standard parameters except the low complexityfilter, which is turned off, and the substitution matrix of BLOSUM62,filters for top 5 hits yielding sequence matches. The nucleotidesequences are translated in all six frames and the frame with no stopcodons in the matching segment of the database sequence is consideredthe potential hit. This is in turn confirmed using the BLAST programtblastx, which translates the antibody sequence in all six frames andcompares those translations to the VBASE nucleotide sequencesdynamically translated in all six frames.

The identities are exact amino acid matches between the antibodysequence and the protein database over the entire length of thesequence. The positives (identities+substitution match) are notidentical but amino acid substitutions guided by the BLOSUM62substitution matrix. If the antibody sequence matches two of thedatabase sequences with same identity, the hit with most positives wouldbe decided to be the matching sequence hit.

Preferred framework sequences for use in the antibodies of thisdisclosure are those that are structurally similar to the frameworksequences used by selected antibodies of this disclosure, e.g., similarto the V_(H) 4-34 framework sequences (SEQ ID NO: 51) and/or the V_(H)3-53 framework sequences (SEQ ID NO: 52) and/or the combined V_(H)3-9/D3-10/JH6b framework sequences (SEQ ID NO: 53) and/or the V_(K) A27framework sequences (SEQ ID NO: 54) and/or the combined V_(K) L6/JK1framework sequences (SEQ ID NO: 55) used by preferred monoclonalantibodies of this disclosure. The V_(H) CDR1, CDR2 and CDR3 sequencesand the V_(K) CDR1, CDR2 and CDR3 sequences, can be grafted ontoframework regions that have the identical sequence as that found in thegermline immunoglobulin gene from which the framework sequence derive orthe CDR sequences can be grafted onto framework regions that contain oneor more mutations as compared to the germline sequences. For example, ithas been found that in certain instances it is beneficial to mutateresidues within the framework regions to maintain or enhance the antigenbinding ability of the antibody (see e.g., U.S. Pat. Nos. 5,530,101;5,585,089; 5,693,762 and 6,180,370 to Queen et al).

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(K) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutation(s) and the effecton antibody binding or other functional property of interest, can beevaluated in in vitro or in vivo assays as described herein and providedin the Examples. Typically conservative modifications (as discussedabove) are introduced. The mutations may be amino acid substitutions,additions or deletions, but are typically substitutions. Moreover,typically no more than one, two, three, four or five residues within aCDR region are altered.

Accordingly, in another embodiment, this disclosure provides isolatedanti-O8E monoclonal antibodies or antigen binding portions thereof,comprising a heavy chain variable region comprising: (a) a V_(H) CDR1region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 11, 12, 13, 14 and 15 or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 11, 12, 13, 14 and 15;(b) a V_(H) CDR2 region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 16, 17, 18, 19 and 20 or an aminoacid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 16, 17,18, 19 and 20; (c) a V_(H) CDR3 region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 21, 22, 23, 24 and 25or an amino acid sequence having one, two, three, four or five aminoacid substitutions, deletions or additions as compared to SEQ ID NOs:21, 22, 23, 24 and 25; (d) a V_(K) CDR1 region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 26, 27, 28,29 and 30 or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to SEQ IDNOs: 26, 27, 28, 29 and 30; (e) a V_(K) CDR2 region comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 31, 32,33, 34 and 35 or an amino acid sequence having one, two, three, four orfive amino acid substitutions, deletions or additions as compared to SEQID NOs: 31, 32, 33, 34 and 35; and (f) a V_(K) CDR3 region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:36, 37, 38, 39 and 40 or an amino acid sequence having one, two, three,four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 36, 37, 38, 39 and 40.

Engineered antibodies of this disclosure include those in whichmodifications have been made to framework residues within V_(H) and/orV_(K), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived.

For example, for 1G11, amino acid residue #71 (within FR3) of V_(H) isan alanine whereas this residue in the corresponding V_(H) 4-34 germlinesequence is a valine. To return the framework region sequences to theirgermline configuration, the somatic mutations can be “backmutated” tothe germline sequence by, for example, site-directed mutagenesis orPCR-mediated mutagenesis (e.g., residue #71 of FR3 of the V_(H) of 1G11can be “backmutated” from alanine to valine). Such “backmutated”antibodies are also intended to be encompassed by this disclosure.

As another example, for 1G11, amino acid residue #81 (within FR3) ofV_(H) is an arginine whereas this residue in the corresponding V_(H)4-34 germline sequence is a lysine. To return the framework regionsequences to their germline configuration, for example, residue #81 ofFR3 of the V_(H) of 1G11 can be “backmutated” from arginine to lysine.Such “backmutated” antibodies are also intended to be encompassed bythis disclosure.

As another example, for 13D12, amino acid residue #83 (within FR3) ofV_(H) is an asparagine whereas this residue in the corresponding V_(H)4-34 germline sequence is a serine. To return the framework regionsequences to their germline configuration, for example, residue #83 ofFR3 of the V_(H) of 13D12 can be “backmutated” from asparagine toserine. Such “backmutated” antibodies are also intended to beencompassed by this disclosure.

As another example, for 2A7, amino acid residue #67 (within FR3) ofV_(H) is a valine whereas this residue in the corresponding V_(H) 3-53germline sequence is an phenylalanine. To return the framework regionsequences to their germline configuration, for example, residue #67 ofFR3 of the V_(H) of 2A7 can be “backmutated” from valine tophenylalanine. Such “backmutated” antibodies are also intended to beencompassed by this disclosure.

As another example, for 2F9, amino acid residue #28 (within FR1) ofV_(H) is a isoleucine whereas this residue in the corresponding V_(H)3-53 germline sequence is a threonine. To return the framework regionsequences to their germline configuration, for example, residue #28 ofFR1 of the V_(H) of 2F9 can be “backmutated” from isoleucine tothreonine. Such “backmutated” antibodies are also intended to beencompassed by this disclosure.

As another example, for 12E1, amino acid residue #23 (within FR1) ofV_(H) is a valine whereas this residue in the corresponding V_(H) 3-9germline sequence is an alanine. To return the framework regionsequences to their germline configuration, for example, residue #23 ofFR1 of the V_(H) of 12E1 can be “backmutated” from valine to alanine.Such “backmutated” antibodies are also intended to be encompassed bythis disclosure.

As another example, for 1G11, amino acid residue #7 (within FR1) ofV_(k) is a phenylalanine whereas this residue in the corresponding V_(k)A27 germline sequence is a serine. To return the framework regionsequences to their germline configuration, for example, residue #7 ofFR1 of the V_(k) of 1G11 can be “backmutated” from phenylalanine toserine. Such “backmutated” antibodies are also intended to beencompassed by this disclosure.

As another example, for 1G11, amino acid residue #47 (within FR2) ofV_(k) is a valine whereas this residue in the corresponding V_(k) A27germline sequence is a leucine. To return the framework region sequencesto their germline configuration, for example, residue #47 of FR2 of theV_(k) of 1G11 can be “backmutated” from valine to leucine. Such“backmutated” antibodies are also intended to be encompassed by thisdisclosure.

Another type of framework modification involves mutating one or moreresidues within the framework region or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

Engineered antibodies of this disclosure also include those in whichmodifications have been made to amino acid residues to increase ordecrease immunogenic responses through amino acid modifications thatalter interaction of a T-cell epitope on the antibody (see e.g., U.S.Pat. Nos. 6,835,550; 6,897,049 and 6,936,249).

In addition or alternative to modifications made within the framework orCDR regions, antibodies of this disclosure may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of this disclosure maybe chemically modified (e.g., one or more chemical moieties can beattached to the antibody) or be modified to alter its glycosylation,again to alter one or more functional properties of the antibody. Eachof these embodiments is described in further detail below. The numberingof residues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has an altered affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the C1 component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered Clq binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551 by Idusogie etal.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351 by Bodmer et al.

In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Thisapproach is described further in PCT Publication WO 00/42072 by Presta.Moreover, the binding sites on human IgG1 for FcγR1. FcγRII, FcγRIII andFcRn have been mapped and variants with improved binding have beendescribed (see Shields, R. L. et al. (2001) J. Biol. Chem.276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334and 339 were shown to improve binding to FcγRIII. Additionally, thefollowing combination mutants were shown to improve FcγRIII binding:T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e. theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of this disclosure to thereby produce an antibodywith altered glycosylation. For example, the cell lines Ms704, Ms705 andMs709 lack the fucosyltransferase gene, FUT8 (alpha (1,6)fucosyltransferase), such that antibodies expressed in the Ms704, Ms705and Ms709 cell lines lack fucose on their carbohydrates. The Ms704,Ms705 and Ms709 FUT8^(−/−) cell lines were created by the targeteddisruption of the FUT8 gene in CHO/DG44 cells using two replacementvectors (see U.S. Patent Publication No. 20040110704 by Yamane et al.and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As anotherexample, EP 1,176,195 by Hanai et al. describes a cell line with afunctionally disrupted FUT8 gene, which encodes a fucosyl transferase,such that antibodies expressed in such a cell line exhibithypofucosylation by reducing or eliminating the alpha 1,6 bond-relatedenzyme. Hanai et al. also describe cell lines which have a low enzymeactivity for adding fucose to the N-acetylglucosamine that binds to theFc region of the antibody or does not have the enzyme activity, forexample the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT PublicationWO 03/035835 by Presta describes a variant CHO cell line, Lec13 cells,with reduced ability to attach fucose to Asn(297)-linked carbohydrates,also resulting in hypofucosylation of antibodies expressed in that hostcell (see also Shields, R. L. et al. (2002) J. Biol. Chem.277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describescell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)—N-acetylglucosaminyltransferase III(GnTIII)) such that antibodies expressed in the engineered cell linesexhibit increased bisecting GlcNac structures which results in increasedADCC activity of the antibodies (see also Umana et al. (1999) Nat.Biotech. 17:176-180). Alternatively, the fucose residues of the antibodymay be cleaved off using a fucosidase enzyme. For example, thefucosidase alpha-L-fucosidase removes fucosyl residues from antibodies(Tarentino, A. L. et al. (1975) Biochem. 14:5516-23).

Another modification of the antibodies herein that is contemplated bythis disclosure is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half life of theantibody. To pegylate an antibody, the antibody or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.Typically, the pegylation is carried out via an acylation reaction or analkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of this disclosure. See for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

Methods of Engineering Antibodies

As discussed above, the anti-O8E antibodies having V_(H) and V_(K)sequences disclosed herein can be used to create new anti-O8E antibodiesby modifying the V_(H) and/or V_(K) sequences or the constant region(s)attached thereto. Thus, in another aspect of this disclosure, thestructural features of an anti-O8E antibody of this disclosure, e.g.1G11, 2A7, 2F9, 12E1 or 13D12, are used to create structurally relatedanti-O8E antibodies that retain at least one functional property of theantibodies of this disclosure, such as binding to human O8E. Forexample, one or more CDR regions of 1G11, 2A7, 2F9, 12E1 or 13D12 ormutations thereof, can be combined recombinantly with known frameworkregions and/or other CDRs to create additional,recombinantly-engineered, anti-O8E antibodies of this disclosure, asdiscussed above. Other types of modifications include those described inthe previous section. The starting material for the engineering methodis one or more of the V_(H) and/or V_(K) sequences provided herein orone or more CDR regions thereof. To create the engineered antibody, itis not necessary to actually prepare (i.e. express as a protein) anantibody having one or more of the V_(H) and/or V_(K) sequences providedherein or one or more CDR regions thereof. Rather, the informationcontained in the sequence(s) is used as the starting material to createa “second generation” sequence(s) derived from the original sequence(s)and then the “second generation” sequence(s) is prepared and expressedas a protein.

Accordingly, in another embodiment, this disclosure provides a methodfor preparing an anti-O8E antibody comprising:

-   -   (a) providing: (i) a heavy chain variable region antibody        sequence comprising a CDR1 sequence selected from the group        consisting of SEQ ID NOs: 11, 12, 13, 14 and 15, a CDR2 sequence        selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19        and 20 and/or a CDR3 sequence selected from the group consisting        of SEQ ID NOs: 21, 22, 23, 24 and 25; and/or (ii) a light chain        variable region antibody sequence comprising a CDR1 sequence        selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29        and 30, a CDR2 sequence selected from the group consisting of        SEQ ID NOs: 31, 32, 33, 34 and 35 and/or a CDR3 sequence        selected from the group consisting of SEQ ID NOs: 36, 37, 38, 39        and 40;    -   (b) altering at least one amino acid residue within the heavy        chain variable region antibody sequence and/or the light chain        variable region antibody sequence to create at least one altered        antibody sequence; and    -   (c) expressing the altered antibody sequence as a protein.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence.

Typically, the antibody encoded by the altered antibody sequence(s) isone that retains one, some or all of the functional properties of theanti-O8E antibodies described herein, which functional propertiesinclude, but are not limited to:

(i) binds to human O8E with a K_(D) of 1×10⁻⁷ M or less;

(ii) binds to human CHO cells transfected with O8E.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples (e.g., flow cytometry, bindingassays).

In certain embodiments of the methods of engineering antibodies of thisdisclosure, mutations can be introduced randomly or selectively alongall or part of an anti-O8E antibody coding sequence and the resultingmodified anti-O8E antibodies can be screened for binding activity and/orother functional properties as described herein. Mutational methods havebeen described in the art. For example, PCT Publication WO 02/092780 byShort describes methods for creating and screening antibody mutationsusing saturation mutagenesis, synthetic ligation assembly or acombination thereof. Alternatively, PCT Publication WO 03/074679 byLazar et al. describes methods of using computational screening methodsto optimize physiochemical properties of antibodies.

Nucleic Acid Molecules Encoding Antibodies of this Disclosure

Another aspect of this disclosure pertains to nucleic acid moleculesthat encode the antibodies of this disclosure. The nucleic acids may bepresent in whole cells, in a cell lysate or in a partially purified orsubstantially pure form. A nucleic acid is “isolated” or “renderedsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, F. Ausubel, et al., ed. (1987) Current Protocols inMolecular Biology, Greene Publishing and Wiley Interscience, New York. Anucleic acid of this disclosure can be, for example, DNA or RNA and mayor may not contain intronic sequences. In a preferred embodiment, thenucleic acid is a cDNA molecule.

Nucleic acids of this disclosure can be obtained using standardmolecular biology techniques. For antibodies expressed by hybridomas(e.g., hybridomas prepared from transgenic mice carrying humanimmunoglobulin genes as described further below), cDNAs encoding thelight and heavy chains of the antibody made by the hybridoma can beobtained by standard PCR amplification or cDNA cloning techniques. Forantibodies obtained from an immunoglobulin gene library (e.g., usingphage display techniques), nucleic acid encoding the antibody can berecovered from the library.

Preferred nucleic acids molecules of this disclosure are those encodingthe VH and VL sequences of the 1G11, 2A7, 2F9, 12E1 or 13D12 monoclonalantibodies. DNA sequences encoding the V_(H) sequences of 1G11, 2A7,2F9, 12E1 and 13D12 are shown in SEQ ID NOs: 41, 42, 43, 44 and 45,respectively. DNA sequences encoding the V_(L) sequences of 1G11, 2A7,2F9, 12E1 and 13D12 are shown in SEQ ID NOs: 46, 47, 48, 49 and 50,respectively.

Once DNA fragments encoding V_(H) and V_(L) segments are obtained, theseDNA fragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a V_(L)- or V_(H)-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most typically isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the V_(H)-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, C_(L). The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabat,E. A., et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification. The light chain constantregion can be a kappa or lambda constant region, but most typically is akappa constant region.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (seee.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature348:552-554).

Production of Monoclonal Antibodies of this Disclosure

Monoclonal antibodies (mAbs) of the present disclosure can be producedby a variety of techniques, including conventional monoclonal antibodymethodology e.g., the standard somatic cell hybridization technique ofKohler and Milstein (1975) Nature 256: 495. Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibody can be employed e.g., viral oroncogenic transformation of B lymphocytes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present disclosure can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the non-human hybridoma of interestand engineered to contain human immunoglobulin sequences using standardmolecular biology techniques. For example, to create a chimericantibody, the murine variable regions can be linked to human constantregions using methods known in the art (see e.g., U.S. Pat. No.4,816,567 to Cabilly et al.). To create a humanized antibody, the murineCDR regions can be inserted into a human framework using methods knownin the art (see e.g., U.S. Pat. No. 5,225,539 to Winter and U.S. Pat.Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

In a preferred embodiment, the antibodies of this disclosure are humanmonoclonal antibodies. Such human monoclonal antibodies directed againstO8E can be generated using transgenic or transchromosomic mice carryingparts of the human immune system rather than the mouse system. Thesetransgenic and transchromosomic mice include mice referred to herein asthe HuMAb Mouse® and KM Mouse®, respectively and are collectivelyreferred to herein as “human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (see e.g., Lonberg, et al.(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or K and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N.(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. 13: 65-93 and Harding, F. andLonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparationand use of HuMab mice and the genomic modifications carried by suchmice, is further described in Taylor, L. et al. (1992) Nucleic AcidsResearch 20:6287-6295; Chen, J. et al. (1993) International Immunology5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. etal. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol.152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, the contents of all of which are hereby specificallyincorporated by reference in their entirety. See further, U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay;U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 toKorman et al.

In another embodiment, human antibodies of this disclosure can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as the “KM Mouse®”, are described in detail in PCT PublicationWO 02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-O8E antibodies of this disclosure. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused; such mice are described in, for example, U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6, 150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-O8E antibodies of this disclosure. For example, mice carrying botha human heavy chain transchromosome and a human light chaintranschromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA97:722-727. As another example, cows carrying human heavy and lightchain transchromosomes have been described in the art (Kuroiwa et al.(2002) Nature Biotechnology 20:889-894) and can be used to raiseanti-O8E antibodies of this disclosure.

Human monoclonal antibodies of this disclosure can also be preparedusing phage display methods for screening libraries of humanimmunoglobulin genes. Such phage display methods for isolating humanantibodies are established in the art. See for example: U.S. Pat. Nos.5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos.5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and6,172,197 to McCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404;6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies of this disclosure can also be preparedusing SCID mice into which human immune cells have been reconstitutedsuch that a human antibody response can be generated upon immunization.Such mice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Immunization of Human Ig Mice

When human Ig mice are used to raise human antibodies of thisdisclosure, such mice can be immunized with a O8E-expressing cell line,a purified or enriched preparation of O8E antigen and/or recombinant O8Eor an O8E fusion protein, as described by Lonberg, N. et al. (1994)Nature 368(6474): 856-859; Fishwild, D. et al. (1996) NatureBiotechnology 14: 845-851; and PCT Publication WO 98/24884 and WO01/14424. Typically, the mice will be 6-16 weeks of age upon the firstimmunization. For example, a purified or recombinant preparation (5-50μg) of O8E antigen can be used to immunize the human Ig miceintraperitoneally.

Detailed procedures to generate fully human monoclonal antibodies to O8Eare described in Example 1 below. Cumulative experience with variousantigens has shown that the transgenic mice respond when initiallyimmunized intraperitoneally (IP) with antigen in complete Freund'sadjuvant, followed by every other week IP immunizations up to a total of6) with antigen in incomplete Freund's adjuvant. However, adjuvantsother than Freund's are also found to be effective. In addition, wholecells in the absence of adjuvant are found to be highly immunogenic. Theimmune response can be monitored over the course of the immunizationprotocol with plasma samples being obtained, for example, byretroorbital bleeds. The plasma can be screened by ELISA and mice withsufficient titers of anti-O8E human immunoglobulin can be used forfusions (as described in Example 1). Mice can be boosted intravenouslywith antigen 3 days before sacrifice and removal of the spleen. It isexpected that 2-3 fusions for each immunization may need to beperformed. Between 6 and 24 mice are typically immunized for eachantigen. Usually both HCo7 and HCo12 strains are used. Generation ofHCo7 and HCo12 mouse strains are described in U.S. Pat. No. 5,770,429and Example 2 of PCT Publication WO 01/09187, respectively. In addition,both HCo7 and HCo12 transgene can be bred together into a single mousehaving two different human heavy chain transgenes (HCo7/HCo12).Alternatively or additionally, the KM mouses strain can be used, asdescribed in PCT Publication WO 02/43478.

Generation of Hybridomas Producing Human Monoclonal Antibodies of thisDisclosure

To generate hybridomas producing human monoclonal antibodies of thisdisclosure, splenocytes and/or lymph node cells from immunized mice canbe isolated and fused to an appropriate immortalized cell line, such asa mouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toone-third the number of Sp2/0 nonsecreting mouse myeloma cells (ATCC,CRL 1581) with 50% PEG. Alternatively, the single cell suspensions ofsplenic lymphocytes from immunized mice can be fused to an equal numberof Sp2/0 mouse myeloma cells using an electric field based electrofusionmethod, using a Cyto Pulse large chamber cell fusion electroporator(Cyto Pulse Sciences, Inc., Glen Burnie, Md.). Cells are plated atapproximately 1×10⁵ cells/well in flat bottom microtiter plate, followedby a two week incubation in selective medium containing 10% fetal bovineserum (Hyclone, Logan, Utah), 10% P388DI (ATCC, CRL TIB-63) conditionedmedium, 3-5% origen (IGEN) in DMEM (Mediatech, CRL 10013, with highglucose, L-glutamine and sodium pyruvate) plus 5 mM HEPES, 0.055 mM2-mercaptoethanol, 50 mg/ml gentamycin and 1×HAT (Sigma, CRL P-7185).After approximately 1-2 weeks, cells can be cultured in medium in whichthe HAT is replaced with HT. Individual wells can then be screened byELISA or FACS for human monoclonal IgM and IgG antibodies. The positiveclones can then be screened for O8E positive antibodies on O8Erecombinant protein by ELISA or on O8E expressing cells, for exampleCHO-O8E transfected cells, by FACS. Once extensive hybridoma growthoccurs, medium can be observed usually after 10-14 days. The antibodysecreting hybridomas can be replated, screened again and if stillpositive for human IgG, the monoclonal antibodies can be subcloned atleast twice by limiting dilution. The stable subclones can then becultured in vitro to generate small amounts of antibody in tissueculture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

Generation of Transfectomas Producing Monoclonal Antibodies of thisDisclosure

Antibodies of this disclosure also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification or cDNA cloning using a hybridoma that expresses theantibody of interest) and the DNAs can be inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. In this context, the term“operatively linked” is intended to mean that an antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the C_(H) segment(s) within the vectorand the V_(K) segment is operatively linked to the C_(L) segment withinthe vector. Additionally or alternatively, the recombinant expressionvector can encode a signal peptide that facilitates secretion of theantibody chain from a host cell. The antibody chain gene can be clonedinto the vector such that the signal peptide is linked in-frame to theamino terminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e. asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of this disclosure carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes,Such regulatory sequences are described, for example, in Goeddel (GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990)). It will be appreciated by those skilled in theart that the design of the expression vector, including the selection ofregulatory sequences, may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP) and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or β-globin promoter. Still further, regulatory elementscomposed of sequences from different sources, such as the SRα promotersystem, which contains sequences from the SV40 early promoter and thelong terminal repeat of human T cell leukemia virus type 1 (Takebe, Y.et al. (1988) Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of this disclosure may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g. origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of this disclosure in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells and most typically mammalian host cells, is the mostpreferred because such eukaryotic cells and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof this disclosure include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another preferred expression system isthe GS gene expression system disclosed in WO 87/04462, WO 89/01036 andEP 338,841. When recombinant expression vectors encoding antibody genesare introduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more typically,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

Characterization of Antibody Binding to Antigen

Antibodies of this disclosure can be tested for binding to O8E by, forexample, flow cytometry. Briefly, O8E-expressing cells are freshlyharvested from tissue culture flasks and a single cell suspensionprepared. O8E-expressing cell suspensions are either stained withprimary antibody directly or after fixation with 1% paraformaldehyde inPBS. Approximately one million cells are resuspended in PBS containing0.5% BSA and 50-200 μg/ml of primary antibody and incubated on ice for30 minutes. The cells are washed twice with PBS containing 0.1% BSA,0.01% NaN₃, resuspended in 100 μl of 1:100 diluted FITC-conjugatedgoat-anti-human IgG (Jackson ImmunoResearch, West Grove, Pa.) andincubated on ice for an additional 30 minutes. The cells are againwashed twice, resuspended in 0.5 ml of wash buffer and analyzed forfluorescent staining on a FACSCalibur cytometer (Becton-Dickinson, SanJose, Calif.).

Alternatively, antibodies of this disclosure can be tested for bindingto O8E by standard ELISA. Briefly, microtiter plates are coated withpurified O8E at 0.25 μg/ml in PBS and then blocked with 5% bovine serumalbumin in PBS. Dilutions of antibody (e.g., dilutions of plasma fromO8E-immunized mice) are added to each well and incubated for 1-2 hoursat 37° C. The plates are washed with PBS/Tween and then incubated withsecondary reagent (e.g., for human antibodies, a goat-anti-human IgGFc-specific polyclonal reagent) conjugated to alkaline phosphatase for 1hour at 37° C. After washing, the plates are developed with pNPPsubstrate (1 mg/ml) and analyzed at OD of 405-650. Typically, mice whichdevelop the highest titers will be used for fusions.

An ELISA or FACS assay, as described above, can also be used to screenfor hybridomas that show positive reactivity with O8E immunogen.Hybridomas that bind with high avidity to O8E are subcloned and furthercharacterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA or FACS), can be chosen formaking a 5-10 vial cell bank stored at −140° C. and for antibodypurification.

To purify anti-O8E antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected anti-O8E monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Competition studies usingunlabeled monoclonal antibodies and biotinylated monoclonal antibodiescan be performed using O8E coated-ELISA plates as described above.Biotinylated mAb binding can be detected with a strep-avidin-alkalinephosphatase probe. Alternatively, competition studies can be performedusing radiolabelled antibody and unlabelled competing antibodies can bedetected in a Scatchard analysis, as further described in the Examplesbelow.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed using reagents specific for antibodies of a particularisotype. For example, to determine the isotype of a human monoclonalantibody, wells of microtiter plates can be coated with 1 μg/ml ofanti-human immunoglobulin overnight at 4° C. After blocking with 1% BSA,the plates are reacted with 1 μg/ml or less of test monoclonalantibodies or purified isotype controls, at ambient temperature for oneto two hours. The wells can then be reacted with either human IgG1 orhuman IgM-specific alkaline phosphatase-conjugated probes. Plates aredeveloped and analyzed as described above.

Anti-O8E human IgGs can be further tested for reactivity with O8Eantigen by Western blotting. Briefly, O8E can be prepared and subjectedto sodium dodecyl sulfate polyacrylamide gel electrophoresis. Afterelectrophoresis, the separated antigens are transferred tonitrocellulose membranes, blocked with 10% fetal calf serum and probedwith the monoclonal antibodies to be tested. Human IgG binding can bedetected using anti-human IgG alkaline phosphatase and developed withBCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

Antibody Physical Properties

The antibodies of the present disclosure may be further characterized bythe various physical properties of the anti-O8E antibodies. Variousassays may be used to detect and/or differentiate different classes ofantibodies based on these physical properties.

In some embodiments, antibodies of the present disclosure may containone or more glycosylation sites in either the light or heavy chainvariable region. The presence of one or more glycosylation sites in thevariable region may result in increased immunogenicity of the antibodyor an alteration of the pK of the antibody due to altered antigenbinding (Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala F A andMorrison S L (2004) J Immunol 172:5489-94; Wallick et al (1988) J ExpMed 168:1099-109; Spiro R G (2002) Glycobiology 12:43 R-56R; Parekh etal (1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol37:697-706). Glycosylation has been known to occur at motifs containingan N-X-S/T sequence. Variable region glycosylation may be tested using aGlycoblot assay, which cleaves the antibody to produce a Fab and thentests for glycosylation using an assay that measures periodate oxidationand Schiff base formation. Alternatively, variable region glycosylationmay be tested using Dionex light chromatography (Dionex-LC), whichcleaves saccharides from a Fab into monosaccharides and analyzes theindividual saccharide content. In some instances, it is preferred tohave an anti-O8E antibody that does not contain variable regionglycosylation. This can be achieved either by selecting antibodies thatdo not contain the glycosylation motif in the variable region or bymutating residues within the glycosylation motif using standardtechniques well known in the art.

In a preferred embodiment, the antibodies of the present disclosure donot contain asparagine isomerism sites. A deamidation or isoasparticacid effect may occur on N-G or D-G sequences, respectively. Thedeamidation or isoaspartic acid effect results in the creation ofisoaspartic acid which decreases the stability of an antibody bycreating a kinked structure off a side chain carboxy terminus ratherthan the main chain. The creation of isoaspartic acid can be measuredusing an iso-quant assay, which uses a reverse-phase HPLC to test forisoaspartic acid.

Each antibody will have a unique isoelectric point (pI), but generallyantibodies will fall in the pH range of between 6 and 9.5. The pI for anIgG1 antibody typically falls within the pH range of 7-9.5 and the pIfor an IgG4 antibody typically falls within the pH range of 6-8.Antibodies may have a pI that is outside this range. Although theeffects are generally unknown, there is speculation that antibodies witha pI outside the normal range may have some unfolding and instabilityunder in vivo conditions. The isoelectric point may be tested using acapillary isoelectric focusing assay, which creates a pH gradient andmay utilize laser focusing for increased accuracy (Janini et al (2002)Electrophoresis 23:1605-11; Ma et al. (2001) Chromatographia 53:S75-89;Hunt et al (1998) J Chromatogr A 800:355-67). In some instances, it ispreferred to have an anti-O8E antibody that contains a pI value thatfalls in the normal range. This can be achieved either by selectingantibodies with a pI in the normal range or by mutating charged surfaceresidues using standard techniques well known in the art.

Each antibody will have a melting temperature that is indicative ofthermal stability (Krishnamurthy R and Manning M C (2002) Curr PharmBiotechnol 3:361-71). A higher thermal stability indicates greateroverall antibody stability in vivo. The melting point of an antibody maybe measure using techniques such as differential scanning calorimetry(Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) ImmunolLett 68:47-52). T_(M1) indicates the temperature of the initialunfolding of the antibody. T_(M2) indicates the temperature of completeunfolding of the antibody. Generally, it is preferred that the T_(M1) ofan antibody of the present disclosure is greater than 60° C., preferablygreater than 65° C., even more preferably greater than 70° C.Alternatively, the thermal stability of an antibody may be measure usingcircular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9).

In a preferred embodiment, antibodies are selected that do not rapidlydegrade. Fragmentation of an anti-O8E antibody may be measured usingcapillary electrophoresis (CE) and MALDI-MS, as is well understood inthe art (Alexander A J and Hughes D E (1995) Anal Chem 67:3626-32).

In another preferred embodiment, antibodies are selected that haveminimal aggregation effects. Aggregation may lead to triggering of anunwanted immune response and/or altered or unfavorable pharmacokineticproperties. Generally, antibodies are acceptable with aggregation of 25%or less, preferably 20% or less, even more preferably 15% or less, evenmore preferably 10% or less and even more preferably 5% or less.Aggregation may be measured by several techniques well known in the art,including size-exclusion column (SEC) high performance liquidchromatography (HPLC) and light scattering to identify monomers, dimers,trimers or multimers.

Immunoconjugates

In another aspect, the present disclosure features an anti-O8E antibodyor a fragment thereof, conjugated to a therapeutic moiety, such as acytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin. Suchconjugates are referred to herein as “immunoconjugates”.Immunoconjugates that include one or more cytotoxins are referred to as“immunotoxins.” A cytotoxin or cytotoxic agent includes any agent thatis detrimental to (e.g., kills) cells. Examples include taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol and puromycin and analogs or homologsthereof. Therapeutic agents also include, for example, antimetabolites(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine(CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin,mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin and anthramycin (AMC)) and anti-mitotic agents(e.g., vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can beconjugated to an antibody of this disclosure include duocarmycins,calicheamicins, maytansines and auristatins and derivatives thereof. Anexample of a calicheamicin antibody conjugate is commercially available(Mylotarg™; Wyeth-Ayerst).

Cytoxins can be conjugated to antibodies of this disclosure using linkertechnology available in the art. Examples of linker types that have beenused to conjugate a cytotoxin to an antibody include, but are notlimited to, hydrazones, thioethers, esters, disulfides andpeptide-containing linkers. A linker can be chosen that is, for example,susceptible to cleavage by low pH within the lysosomal compartment orsusceptible to cleavage by proteases, such as proteases preferentiallyexpressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito, G. et al.(2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, P. A. et al. (2003)Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell3:207-212; Allen, T. M. (2002) Nat. Rev. Cancer 2:750-763; Pastan, I.and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3:1089-1091;Senter, P. D. and Springer, C. J. (2001) Adv. Drug Deliv. Rev.53:247-264.

Antibodies of the present disclosure also can be conjugated to aradioactive isotope to generate cytotoxic radiopharmaceuticals, alsoreferred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine¹³¹, iodine¹²⁵,indium¹¹¹, yttrium⁹⁰ and lutetium¹⁷⁷. Method for preparingradioimmunconjugates are established in the art. Examples ofradioimmunoconjugates are commercially available, including Zevalin™(IDEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals) and similarmethods can be used to prepare radioimmunoconjugates using theantibodies of this disclosure.

The antibody conjugates of this disclosure can be used to modify a givenbiological response and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”) or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results and Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985) and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Bispecific Molecules

In another aspect, the present disclosure features bispecific moleculescomprising an anti-O8E antibody or a fragment thereof, of thisdisclosure. An antibody of this disclosure or antigen-binding portionsthereof, can be derivatized or linked to another functional molecule,e.g., another peptide or protein (e.g., another antibody or ligand for areceptor) to generate a bispecific molecule that binds to at least twodifferent binding sites or target molecules. The antibody of thisdisclosure may in fact be derivatized or linked to more than one otherfunctional molecule to generate multispecific molecules that bind tomore than two different binding sites and/or target molecules; suchmultispecific molecules are also intended to be encompassed by the term“bispecific molecule” as used herein. To create a bispecific molecule ofthis disclosure, an antibody of this disclosure can be functionallylinked (e.g., by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other binding molecules, suchas another antibody, antibody fragment, peptide or binding mimetic, suchthat a bispecific molecule results.

Accordingly, the present disclosure includes bispecific moleculescomprising at least one first binding specificity for O8E and a secondbinding specificity for a second target epitope. In a particularembodiment of this disclosure, the second target epitope is an Fcreceptor, e.g., human FcγRI (CD64) or a human Fcα receptor (CD89).Therefore, this disclosure includes bispecific molecules capable ofbinding both to FcγR or FcαR expressing effector cells (e.g., monocytes,macrophages or polymorphonuclear cells (PMNs)) and to target cellsexpressing O8E. These bispecific molecules target O8E expressing cellsto effector cell and trigger Fc receptor-mediated effector cellactivities, such as phagocytosis of an O8E expressing cells, antibodydependent cell-mediated cytotoxicity (ADCC), cytokine release orgeneration of superoxide anion.

In an embodiment of this disclosure in which the bispecific molecule ismultispecific, the molecule can further include a third bindingspecificity, in addition to an anti-Fc binding specificity and ananti-O8E binding specificity. In one embodiment, the third bindingspecificity is an anti-enhancement factor (EF) portion, e.g., a moleculewhich binds to a surface protein involved in cytotoxic activity andthereby increases the immune response against the target cell. The“anti-enhancement factor portion” can be an antibody, functionalantibody fragment or a ligand that binds to a given molecule, e.g., anantigen or a receptor and thereby results in an enhancement of theeffect of the binding determinants for the F_(c) receptor or target cellantigen. The “anti-enhancement factor portion” can bind an F_(c)receptor or a target cell antigen. Alternatively, the anti-enhancementfactor portion can bind to an entity that is different from the entityto which the first and second binding specificities bind. For example,the anti-enhancement factor portion can bind a cytotoxic T-cell (e.g.via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell thatresults in an increased immune response against the target cell).

In one embodiment, the bispecific molecules of this disclosure compriseas a binding specificity at least one antibody or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, Fd, dAb or a singlechain Fv. The antibody may also be a light chain or heavy chain dimer orany minimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778, the contents ofwhich is expressly incorporated by reference.

In one embodiment, the binding specificity for an Fcγ receptor isprovided by a monoclonal antibody, the binding of which is not blockedby human immunoglobulin G (IgG). As used herein, the term “IgG receptor”refers to any of the eight γ-chain genes located on chromosome 1. Thesegenes encode a total of twelve transmembrane or soluble receptorisoforms which are grouped into three Fcγ receptor classes: FcγR (CD64),FcγRII (CD32) and FcγRIII (CD16). In one preferred embodiment, the Fcγreceptor a human high affinity FcγRI. The human FcγRI is a 72 kDamolecule, which shows high affinity for monomeric IgG (10⁸-10⁹ M⁻¹)

The production and characterization of certain preferred anti-Fcγmonoclonal antibodies are described by Fanger et al. in PCT PublicationWO 88/00052 and in U.S. Pat. No. 4,954,617, the teachings of which arefully incorporated by reference herein. These antibodies bind to anepitope of FcγRI, FcγRII or FcγRIII at a site which is distinct from theFcγ binding site of the receptor and, thus, their binding is not blockedsubstantially by physiological levels of IgG. Specific anti-FcγRIantibodies useful in this disclosure are mAb 22, mAb 32, mAb 44, mAb 62and mAb 197. The hybridoma producing mAb 32 is available from theAmerican Type Culture Collection, ATCC Accession No. HB9469. In otherembodiments, the anti-Fcγ receptor antibody is a humanized form ofmonoclonal antibody 22 (H22). The production and characterization of theH22 antibody is described in Graziano, R. F. et al. (1995) J. Immunol155 (10): 4996-5002 and PCT Publication WO 94/10332. The H22 antibodyproducing cell line was deposited at the American Type CultureCollection under the designation HA022CL1 and has the accession no. CRL11177.

In still other preferred embodiments, the binding specificity for an Fcreceptor is provided by an antibody that binds to a human IgA receptor,e.g., an Fc-alpha receptor (FcαRI (CD89)), the binding of which istypically not blocked by human immunoglobulin A (IgA). The term “IgAreceptor” is intended to include the gene product of one α-gene (FcαRI)located on chromosome 19. This gene is known to encode severalalternatively spliced transmembrane isoforms of 55 to 110 kDa. FcαRI(CD89) is constitutively expressed on monocytes/macrophages,eosinophilic and neutrophilic granulocytes, but not on non-effector cellpopulations. FcαRI has medium affinity (≈5×10⁷ M⁻¹) for both IgA1 andIgA2, which is increased upon exposure to cytokines such as G-CSF orGM-CSF (Morton, H. C. et al. (1996) Critical Reviews in Immunology16:423-440). Four FcαRI-specific monoclonal antibodies, identified asA3, A59, A62 and A77, which bind FcαRI outside the IgA ligand bindingdomain, have been described (Monteiro, R. C. et al. (1992) J. Immunol.148:1764).

FcαRI and FcγRI are preferred trigger receptors for use in thebispecific molecules of this disclosure because they are (1) expressedprimarily on immune effector cells, e.g., monocytes, PMNs, macrophagesand dendritic cells; (2) expressed at high levels (e.g., 5,000-100,000per cell); (3) mediators of cytotoxic activities (e.g., ADCC,phagocytosis); (4) mediate enhanced antigen presentation of antigens,including self-antigens, targeted to them.

While human monoclonal antibodies are preferred, other antibodies thatcan be employed in the bispecific molecules of this disclosure include,e.g., murine, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present disclosure can be prepared byconjugating the constituent binding specificities, e.g., the anti-FcRand anti-O8E binding specificities, using methods known in the art. Forexample, each binding specificity of the bispecific molecule can begenerated separately and then conjugated to one another. When thebinding specificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyidithio)propionate (SPDP) andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83) and Glennie et al.(1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, typically one,prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand x Fab fusion protein. A bispecific molecule ofthis disclosure can be a single chain molecule comprising one singlechain antibody and a binding determinant or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growthinhibition) or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a 7 counter or a scintillationcounter or by autoradiography.

Pharmaceutical Compositions

In another aspect, the present disclosure provides a composition, e.g.,a pharmaceutical composition, containing one or a combination ofmonoclonal antibodies or antigen-binding portion(s) thereof, of thepresent disclosure, formulated together with a pharmaceuticallyacceptable carrier. Such compositions may include one or a combinationof (e.g., two or more different) antibodies or immunoconjugates orbispecific molecules of this disclosure. For example, a pharmaceuticalcomposition of this disclosure can comprise a combination of antibodies(or immunoconjugates or bispecifics) that bind to different epitopes onthe target antigen or that have complementary activities.

Pharmaceutical compositions of this disclosure also can be administeredin combination therapy, i.e. combined with other agents. For example,the combination therapy can include an anti-O8E antibody of the presentdisclosure combined with at least one other anti-inflammatory orimmunosuppressant agent. Examples of therapeutic agents that can be usedin combination therapy are described in greater detail below in thesection on uses of the antibodies of this disclosure.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like that arephysiologically compatible. Typically, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e. antibody,immunoconjugate or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds of this disclosure may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of this disclosure also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of this disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol and the like) and suitable mixtures thereof,vegetable oils, such as olive oil and injectable organic esters, such asethyl oleate. Proper fluidity can be maintained, for example, by the useof coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthis disclosure is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol and liquid polyethylene glycol andthe like) and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, typically from about0.1 percent to about 70 percent, most typically from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of this disclosure are dictated by anddirectly dependent on (a) the unique characteristics of the activecompound and the particular therapeutic effect to be achieved and (b)the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg and more usually 0.01 to 25 mg/kg, of the host body weight.For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or withinthe range of 1-10 mg/kg. Higher dosages, e.g., 15 mg/kg body weight, 20mg/kg body weight or 25 mg/kg body weight can be used as needed. Anexemplary treatment regime entails administration once per week, onceevery two weeks, once every three weeks, once every four weeks, once amonth, once every 3 months or once every three to 6 months. Particulardosage regimens for an anti-O8E antibody of this disclosure include 1mg/kg body weight or 3 mg/kg body weight via intravenous administration,with the antibody being given using one of the following dosingschedules: (i) every four weeks for six dosages, then every threemonths; (ii) every three weeks; (iii) 3 mg/kg body weight once followedby 1 mg/kg body weight every three weeks.

In some methods, two or more anti-O8E monoclonal antibodies of thisdisclosure with different binding specificities are administeredsimultaneously, in which case the dosage of each antibody administeredfalls within the ranges indicated. Antibody is usually administered onmultiple occasions. Intervals between single dosages can be, forexample, weekly, monthly, every three months or yearly. Intervals canalso be irregular as indicated by measuring blood levels of antibody tothe target antigen in the patient. In some methods, dosage is adjustedto achieve a plasma antibody concentration of about 1-1000 μg/ml and insome methods about 25-300 μg/ml.

In other methods, one or more anti-O8E monoclonal antibody of thisdisclosure are administered simultaneously with an antibody havingdistinct binding specificity such as, for example, anti-CTLA-4 and/oranti-PD-1, in which case the dosage of each antibody administered fallswithin the ranges indicated.

Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human antibodies show the longest half life,followed by humanized antibodies, chimeric antibodies and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated and typically until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present disclosure may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentdisclosure employed or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treatedand like factors well known in the medical arts.

A “therapeutically effective dosage” of an anti-O8E antibody of thisdisclosure typically results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods or a prevention of impairment or disability due to the diseaseaffliction. For example, for the treatment of O8E+ tumors, a“therapeutically effective dosage” typically inhibits cell growth ortumor growth by at least about 20%, more typically by at least about40%, even more typically by at least about 60% and still more typicallyby at least about 80% relative to untreated subjects. The ability of acompound to inhibit tumor growth can be evaluated in an animal modelsystem predictive of efficacy in human tumors. Alternatively, thisproperty of a composition can be evaluated by examining the ability ofthe compound to inhibit, such inhibition in vitro by assays known to theskilled practitioner. A therapeutically effective amount of atherapeutic compound can decrease tumor size or otherwise amelioratesymptoms in a subject. One of ordinary skill in the art would be able todetermine such amounts based on such factors as the subject's size, theseverity of the subject's symptoms and the particular composition orroute of administration selected.

A composition of the present disclosure can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for antibodies of thisdisclosure include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, an antibody of this disclosure can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of this disclosure can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.No. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or4,596,556. Examples of well-known implants and modules useful in thepresent disclosure include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems and modules are known to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of thisdisclosure can be formulated to ensure proper distribution in vivo. Forexample, the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of this disclosurecross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K.Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

Uses and Methods of this Disclosure

The antibodies, particularly the human antibodies, antibody compositionsand methods of the present disclosure have numerous in vitro and in vivodiagnostic and therapeutic utilities involving, for example, detectionof O8E, treatment of cancer or enhancement of immune response byblockade of O8E. In a preferred embodiment, the antibodies of thepresent disclosure are human antibodies. For example, these moleculescan be administered to cells in culture, in vitro or ex vivo or to humansubjects, e.g., in vivo, to treat, prevent and to diagnose a variety ofdisorders or to enhance immunity in a variety of situations,

-   As used herein, the term “subject” is intended to include human and    non-human animals. The term “non-human animals” includes all    vertebrates, e.g., mammals and non-mammals, such as non-human    primates, sheep, dogs, cats, cows, horses, chickens, amphibians and    reptiles. Preferred subjects include human patients having disorders    associated with O8E expression or in need of enhancement of an    immune response. The methods are particularly suitable for treating    human patients having a disorder associated with aberrant O8E    expression. The methods are also particularly suitable for treating    human patients having a disorder that can be treated by augmenting    the T-cell mediated immune response. To achieve antigen-specific    enhancement of immunity, the anti-O8E antibodies can be administered    together with an antigen of interest. When antibodies to O8E are    administered together with another agent, the two can be    administered in either order or simultaneously.

Given the specific binding of the antibodies of this disclosure for O8E,the antibodies of this disclosure can be used to specifically detect O8Eexpression on the surface of cells and, moreover, can be used to purifyO8E via immunoaffinity purification.

Cancer

O8E is expressed in a variety of human cancers, including breast cellcarcinomas, metastatic breast cancers, ovarian cell carcinomas,metastatic ovarian cancers and renal cell carcinomas (Tringler et al.(2005) Clinical Cancer Res. 11:1842-48; Salceda et al. (2005) Exp CellRes. 306:128-41; Tringler et al. (2006) Gynecol Oncol. 100:44-52;Krambeck et al. (2006) Proc Natl Acad Sci USA 103:10391-6; Chen et al.(2006) Kidney Int. Epub; Sun et al. (2006) Lung Cancer 53:143-51;Bignotti et al. (2006) Gynecol Oncol. 103:405-16; Kryczek et al. (2006)J Exp Med 203:871-81; Simon et al. (2006) Cancer Res. 66:1570-5). Ananti-O8E antibody may be used alone to inhibit the growth of canceroustumors. Alternatively, an anti-08E antibody may be used in conjunctionwith other immunogenic agents, standard cancer treatments or otherantibodies, as described below.

The B and T lymphocyte attenuator (BTLA) was found to be the receptorfor O8E and has an inhibitory effect on immune responses, similar tocytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1)(Carreno and Collins (2003) Trends Immunol 24:524-7). O8E functions bynegatively regulating T cell immunity by the inhibition of T-cellproliferation, cytokine production and cell cycle production (Choi etal. (2003) J Immunol. 171:4650-4). An O8E-Ig fusion protein inhibitsT-cell activation, whereas blockade of O8E by antibodies can enhance theimmune response in the patient (Sica et al.(2003) Immunity 18:849-61).

In one aspect, the present disclosure relates to treatment of a subjectin vivo using an anti-08E antibody such that growth of cancerous tumorsis inhibited. An anti-O8E antibody may be used alone to inhibit thegrowth of cancerous tumors. Alternatively, an anti-O8E antibody may beused in conjunction with other immunogenic agents, standard cancertreatments or other antibodies, as described below.

Accordingly, in one embodiment, this disclosure provides a method ofinhibiting growth of tumor cells in a subject, comprising administeringto the subject a therapeutically effective amount of an anti-O8Eantibody or antigen-binding portion thereof. Preferably, the antibody isa human anti-O8E antibody (such as any of the human anti-human O8Eantibodies described herein). Additionally or alternatively, theantibody may be a chimeric or humanized anti-O8E antibody.

Preferred cancers whose growth may be inhibited using the antibodies ofthis disclosure include cancers typically responsive to immunotherapy.Non-limiting examples of preferred cancers for treatment include breastcancer (e.g., breast cell carcinoma), ovarian cancer (e.g., ovarian cellcarcinoma) and renal cell carcinoma (RCC). Examples of other cancersthat may be treated using the methods of this disclosure includemelanoma (e.g., metastatic malignant melanoma), prostate cancer, coloncancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, braintumors, chronic or acute leukemias including acute myeloid leukemia,chronic myeloid leukemia, acute lymphoblastic leukemia, chroniclymphocytic leukemia, lymphomas (e.g., Hodgkin's and non-Hodgkin'slymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma)nasopharangeal carcinomas, cancer of the head or neck, cutaneous orintraocular malignant melanoma, uterine cancer, rectal cancer, cancer ofthe anal region, stomach cancer, testicular cancer, uterine cancer,carcinoma of the fallopian tubes, carcinoma of the endometrium,carcinoma of the cervix, carcinoma of the vagina, carcinoma of thevulva, cancer of the esophagus, cancer of the small intestine, cancer ofthe endocrine system, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the adbreast gland, sarcoma of soft tissue,cancer of the urethra, cancer of the penis, solid tumors of childhood,cancer of the bladder, cancer of the kidney or ureter, carcinoma of thebreast pelvis, neoplasm of the central nervous system (CNS), tumorangiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma,Kaposi's sarcoma, epidermoid cancer, squamous cell cancer,environmentally induced cancers including those induced by asbestos,e.g., mesothelioma and combinations of said cancers.

Optionally, antibodies to O8E can be combined with an immunogenic agent,such as cancerous cells, purified tumor antigens (including recombinantproteins, peptides and carbohydrate molecules), cells and cellstransfected with genes encoding immune stimulating cytokines (He et al,J. Immunol. 173:4919-28 (2004)). Non-limiting examples of tumor vaccinesthat can be used include peptides of melanoma antigens, such as peptidesof gp100, MAGE antigens, Trp-2, MARTI and/or tyrosinase or tumor cellstransfected to express the cytokine GM-CSF.

In humans, some tumors have been shown to be immunogenic such asmelanomas. It is anticipated that by raising the threshold of T cellactivation by O8E blockade, tumors may be activated in responses in thehost.

O8E blockade is likely to be most effective when combined with avaccination protocol. Many experimental strategies for vaccinationagainst tumors have been devised (see, Rosenberg, “Development of CancerVaccines” ASCO Educational Book Spring: 60-62 (2000); Logothetis, ASCOEducational Book Spring: 300-302 (2000); Khayat, ASCO Educational BookSpring: 414-428 (2000); Foon, ASCO Educational Book Spring: 730-738(2000); see also Restifo and Sznol, Cancer Vaccines, Ch. 61, pp.3023-3043 in DeVita et al. (ed.) Cancer: Principles and Practice ofOncology, Fifth Edition (1997)). In one of these strategies, a vaccineis prepared using autologous or allogeneic tumor cells. Typically, thesecellular vaccines are most effective when the tumor cells are transducedto express GM-CSF. GM-CSF has been shown to be a potent activator ofantigen presentation for tumor vaccination (Dranoff et al. Proc. Natl.Acad. Sci. U.S.A. 90: 3539-43 (1993)).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, Immunity 10:281-7 (1999)). In many cases, thesetumor specific antigens are differentiation antigens expressed in thetumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. O8E blockade may be used in conjunction witha collection of recombinant proteins and/or peptides expressed in atumor in order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self antigens andare therefore tolerant to them. The tumor antigen may also include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim et al., Science266:2011-2013 (1994)). (These somatic tissues may be protected fromimmune attack by various means). Tumor antigen may also be“neo-antigens” expressed in cancer cells because of somatic mutationsthat alter protein sequence or create fusion proteins between twounrelated sequences (i.e. bcr-abl in the Philadelphia chromosome) oridiotype from B cell tumors.

Other tumor vaccines may include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which may be used in conjunction with O8Eblockade is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot andSrivastava Science 269:1585-1588 (1995)); Tamura et al. Science278:117-120 (1997)).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle, F. et al. (1998) Nature Medicine 4: 328-332). DCsmay also be transduced by genetic means to express these tumor antigensas well. DCs have also been fused directly to tumor cells for thepurposes of immunization (Kugler, A. et al. (2000) Nature Medicine6:332-336). As a method of vaccination, DC immunization may beeffectively combined with PD-1 blockade to activate more potentanti-tumor responses.

O8E blockade may also be combined with standard cancer treatments. O8Eblockade may be effectively combined with chemotherapeutic regimes. Inthese instances, it may be possible to reduce the dose ofchemotherapeutic reagent administered (Mokyr, M. et al. (1998) CancerResearch 58: 5301-5304). An example of such a combination is an anti-O8Eantibody in combination with decarbazine for the treatment of variouscancers. Another example of such a combination is an anti-O8E antibodyin combination with interleukin-2 (IL-2) for the treatment of variouscancers. The scientific rationale behind the combined use of O8Eblockade and chemotherapy is that cell death, that is a consequence ofthe cytotoxic action of most chemotherapeutic compounds, should resultin increased levels of tumor antigen in the antigen presentationpathway. Other combination therapies that may result in synergy with O8Eblockade through cell death are radiation, surgery and hormonedeprivation. Each of these protocols creates a source of tumor antigenin the host. Angiogenesis inhibitors may also be combined with O8Eblockade. Inhibition of angiogenesis leads to tumor cell death which mayfeed tumor antigen into host antigen presentation pathways.

O8E blocking antibodies can also be used in combination with bispecificantibodies that target Fc alpha or Fc gamma receptor-expressingeffectors cells to tumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and5,837,243). Bispecific antibodies can be used to target two separateantigens. For example anti-Fc receptor/anti tumor antigen (e.g.,Her-2/neu) bispecific antibodies have been used to target macrophages tosites of tumor. This targeting may more effectively activate tumorspecific responses. The T cell arm of these responses would by augmentedby the use of O8E blockade. Alternatively, antigen may be delivereddirectly to DCs by the use of bispecific antibodies which bind to tumorantigen and a dendritic cell specific cell surface marker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-beta (Kehrl, J. et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard, M. & O'Garra, A. (1992) Immunology Today 13:198-200) and Fas ligand (Hahne, M. et al. (1996) Science 274:1363-1365). Antibodies to each of these entities may be used incombination with anti-PD-1 to counteract the effects of theimmunosuppressive agent and favor tumor immune responses by the host.

Other antibodies which may be used to activate host immuneresponsiveness can be used in combination with anti-O8E. These includemolecules on the surface of dendritic cells which activate DC functionand antigen presentation. Anti-CD40 antibodies are able to substituteeffectively for T cell helper activity (Ridge, J. et al. (1998) Nature393: 474-478) and can be used in conjunction with O8E antibodies.Activating antibodies to T cell costimulatory molecules such as CTLA-4(e.g., U.S. Pat. No. 5,811,097), OX-40 (Weinberg, A. et al. (2000)Immunol 164: 2160-2169), 4-1BB (Melero, I. et al. (1997) Nature Medicine3: 682-685 (1997), PD-1 (del Rio et al. (2005) Eur J Immunol.35:3545-60) and ICOS (Hutloff, A. et al. (1999) Nature 397: 262-266) mayalso provide for increased levels of T cell activation.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses. O8E blockade can be used to increase theeffectiveness of the donor engrafted tumor specific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to antigen-specific Tcells against tumor (Greenberg, R. & Riddell, S. (1999) Science 285:546-51). These methods may also be used to activate T cell responses toinfectious agents such as CMV. Ex vivo activation in the presence ofanti-O8E antibodies may be expected to increase the frequency andactivity of the adoptively transferred T cells.

Given the expression of O8E on various tumor cells, the humanantibodies, antibody compositions and methods of the present disclosurecan be used to treat a subject with a tumorigenic disorder, e.g., adisorder characterized by the presence of tumor cells expressing O8Eincluding, for example, breast cancer (e.g., breast cell carcinoma),ovarian cancer (e.g., ovarian cell carcinoma), and renal cancer.Examples of other cancers that may be treated using the methods of theinstant disclosure include melanoma (e.g., metastatic malignantmelanoma), prostate cancer, colon cancer and lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular malignant melanoma, uterine cancer, rectal cancer, cancer ofthe anal region, stomach cancer, testicular cancer, uterine cancer,carcinoma of the fallopian tubes, carcinoma of the endometrium,carcinoma of the cervix, carcinoma of the vagina, carcinoma of thevulva, Hodgkin's Disease, non-Hodgkin's lymphoma, acute lymphocyticleukemia (ALL), chronic lymphocytic leukemia (CLL), Burkitt's lymphoma,anaplastic large-cell lymphomas (ALCL), multiple myeloma, cutaneousT-cell lymphomas, nodular small cleaved-cell lymphomas, lymphocyticlymphomas, peripheral T-cell lymphomas, Lennert's lymphomas,immunoblastic lymphomas, T-cell leukemia/lymphomas (ATLL), adult T-cellleukemia (T-ALL), entroblastic/centrocytic (cb/cc) follicular lymphomascancers, diffuse large cell lymphomas of B lineage, angioimmunoblasticlymphadenopathy (AILD)-like T cell lymphoma, HIV associated body cavitybased lymphomas, embryonal carcinomas, undifferentiated carcinomas ofthe rhino-pharynx (e.g., Schmincke's tumor), Castleman's disease,Kaposi's Sarcoma, multiple myeloma, Waldenstrom's macroglobulinemia andother B-cell lymphomas, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the urethra, cancer of the penis, chronic oracute leukemias including acute myeloid leukemia, chronic myeloidleukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia,solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder,cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasmof the central nervous system (CNS), primary CNS lymphoma, glioblastoma,brain tumors, nasopharangeal carcinomas, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers. The present disclosure is also useful fortreatment of metastatic cancers.

Accordingly, in one embodiment, this disclosure provides a method ofinhibiting growth of tumor cells in a subject, comprising administeringto the subject a therapeutically effective amount of an anti-O8Eantibody or antigen-binding portion thereof. Typically, the antibody isa human anti-O8E antibody (such as any of the human anti-human O8Eantibodies described herein). Additionally or alternatively, theantibody may be a chimeric or humanized anti-O8E antibody.

Infectious Diseases

Other methods of this disclosure are used to treat patients that havebeen exposed to particular toxins or pathogens. Accordingly, anotheraspect of this disclosure provides a method of treating an infectiousdisease in a subject comprising administering to the subject an anti-O8Eantibody or antigen-binding portion thereof, such that the subject istreated for the infectious disease. Preferably, the antibody is a humananti-human O8E antibody (such as any of the human anti-O8E antibodiesdescribed herein). Additionally or alternatively, the antibody can be achimeric or humanized antibody.

Similar to its application to tumors as discussed above, antibodymediated O8E blockade can be used alone or as an adjuvant, incombination with vaccines, to stimulate the immune response topathogens, toxins and self-antigens. Examples of pathogens for whichthis therapeutic approach may be particularly useful, include pathogensfor which there is currently no effective vaccine or pathogens for whichconventional vaccines are less than completely effective. These include,but are not limited to HIV, Hepatitis (A, B, & C), Influenza, Herpes,Giardia, Malaria, Leishmania, Staphylococcus aureus, PseudomonasAeruginosa. PD-1 blockade is particularly useful against establishedinfections by agents such as HIV that present altered antigens over thecourse of the infections. These novel epitopes are recognized as foreignat the time of anti-human O8E administration, thus provoking a strong Tcell response that is not dampened by negative signals through O8E.

Some examples of pathogenic viruses causing infections treatable bymethods of this disclosure include HIV, hepatitis (A, B or C), herpesvirus (e.g., VZV, HSV-1, HAV-6, HSV-II and CMV, Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, cornovirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable bymethods of this disclosure include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand conococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis and Lymes disease bacteria.

Some examples of pathogenic fungi causing infections treatable bymethods of this disclosure include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bymethods of this disclosure include Entamoeba histolytica, Balantidiumcoli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondi, Nippostrongylus brasiliensis.

In all of the above methods, O8E blockade can be combined with otherforms of immunotherapy such as cytokine treatment (e.g., interferons,GM-CSF, G-CSF, IL-2) or bispecific antibody therapy, which provides forenhanced presentation of tumor antigens (see, e.g., Holliger (1993)Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure2:1121-1123).

Autoimmune Reactions

Anti-O8E antibodies may provoke and amplify autoimmune responses.Indeed, induction of anti-tumor responses using tumor cell and peptidevaccines reveals that many anti-tumor responses involve anti-selfreactivities (depigmentation observed in anti-CTLA-4+GM-CSF-modified B16melanoma in van Elsas et al. supra; depigmentation in Trp-2 vaccinatedmice (Overwijk, W. et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:2982-2987); autoimmune prostatitis evoked by TRAMP tumor cell vaccines(Hurwitz, A. (2000) supra), melanoma peptide antigen vaccination andvitilago observed in human clinical trials (Rosenberg, S A and White, DE (1996) J. Immunother Emphasis Tumor Immunol 19 (1): 81-4).

Therefore, it is possible to consider using anti-O8E blockade inconjunction with various self proteins in order to devise vaccinationprotocols to efficiently generate immune responses against these selfproteins for disease treatment. For example, Alzheimers disease involvesinappropriate accumulation of Aβ peptide in amyloid deposits in thebrain; antibody responses against amyloid are able to clear theseamyloid deposits (Schenk et al., (1999) Nature 400: 173-177).

Other self proteins may also be used as targets such as IgE for thetreatment of allergy and asthma and TNFα for rheumatoid arthritis.Finally, antibody responses to various hormones may be induced by theuse of anti-O8E antibody. Neutralizing antibody responses toreproductive hormones may be used for contraception. Neutralizingantibody response to hormones and other soluble factors that arerequired for the growth of particular tumors may also be considered aspossible vaccination targets.

Analogous methods as described above for the use of anti-O8E antibodycan be used for induction of therapeutic autoimmune responses to treatpatients having an inappropriate accumulation of other self-antigens,such as amyloid deposits, including Aβ in Alzheimer's disease, cytokinessuch as TNFα and IgE.

Vaccines

Anti-O8E antibodies may be used to stimulate antigen-specific immuneresponses by coadministration of an anti-O8E antibody with an antigen ofinterest (e.g., a vaccine). Accordingly, in another aspect thisdisclosure provides a method of enhancing an immune response to anantigen in a subject, comprising administering to the subject: (i) theantigen; and (ii) an anti-O8E antibody or antigen-binding portionthereof, such that an immune response to the antigen in the subject isenhanced. Preferably, the antibody is a human anti-human O8E antibody(such as any of the human anti-O8E antibodies described herein).Additionally or alternatively, the antibody can be a chimeric orhumanized antibody. The antigen can be, for example, a tumor antigen, aviral antigen, a bacterial antigen or an antigen from a pathogen.Non-limiting examples of such antigens include those discussed in thesections above, such as the tumor antigens (or tumor vaccines) discussedabove or antigens from the viruses, bacteria or other pathogensdescribed above.

Suitable routes of administering the antibody compositions (e.g., humanmonoclonal antibodies, multispecific and bispecific molecules andimmunoconjugates) of this disclosure in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

As previously described, human anti-O8E antibodies of this disclosurecan be co-administered with one or other more therapeutic agents, e.g.,a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Theantibody can be linked to the agent (as an immunocomplex) or can beadministered separate from the agent. In the latter case (separateadministration), the antibody can be administered before, after orconcurrently with the agent or can be co-administered with other knowntherapies, e.g., an anti-cancer therapy, e.g., radiation. Suchtherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine,chlorambucil, decarbazine and cyclophosphamide hydroxyurea which, bythemselves, are only effective at levels which are toxic or subtoxic toa patient. Cisplatin is intravenously administered as a 100 mg/dose onceevery four weeks and adriamycin is intravenously administered as a 60-75mg/ml dose once every 21 days. Co-administration of the human anti-O8Eantibodies or antigen binding fragments thereof, of the presentdisclosure with chemotherapeutic agents provides two anti-cancer agentswhich operate via different mechanisms which yield a cytotoxic effect tohuman tumor cells. Such co-administration can solve problems due todevelopment of resistance to drugs or a change in the antigenicity ofthe tumor cells which would render them unreactive with the antibody.

Also within the scope of the present disclosure are kits comprising theantibody compositions of this disclosure (e.g., human antibodies,bispecific or multispecific molecules or immunoconjugates) andinstructions for use. The kit can further contain a least one additionalreagent or one or more additional human antibodies of this disclosure(e.g., a human antibody having a complementary activity which binds toan epitope in O8E antigen distinct from the first human antibody). Kitstypically include a label indicating the intended use of the contents ofthe kit. The term label includes any writing or recorded materialsupplied on or with the kit or which otherwise accompanies the kit.

Combination Therapy

In one embodiment, the present disclosure provides a method for treatinga hyperproliferative disease, comprising administering an O8E antibodyand a CTLA-4 and/or PD-1 antibody to a subject. In further embodiments,the anti-O8E antibody is administered at a subtherapeutic dose, theanti-CTLA-4 and/or PD-1 antibody is administered at a subtherapeuticdose or both are administered at a subtherapeutic dose. In anotherembodiment, the present disclosure provides a method for altering anadverse event associated with treatment of a hyperproliferative diseasewith an immunostimulatory agent, comprising administering an anti-O8Eantibody and a subtherapeutic dose of anti-CTLA-4 and/or anti-PD-1antibody to a subject. In certain embodiments, the subject is human. Incertain embodiments, the anti-CTLA-4 antibody is human sequencemonoclonal antibody 10D1 and the anti-PD-1 antibody is human sequencemonoclonal antibody, such as 17D8, 2D3, 4H1, 5C4 and 4A11. Humansequence monoclonal antibody 10D1 has been isolated and structurallycharacterized, as described in U.S. Pat. No. 6,984,720. Human sequencemonoclonal antibodies 17D8, 2D3, 4H1, 5C4 and 4A11 have been isolatedand structurally characterized, as described in U.S. Provisional PatentNo. 60/679,466.

The anti-O8E, anti-CTLA-4 antibody and anti-PD-1 monoclonal antibodies(mAbs) and the human sequence antibodies of this disclosure can beproduced by a variety of techniques, including conventional monoclonalantibody methodology, e.g., the standard somatic cell hybridizationtechnique of Kohler and Milstein (1975) Nature 256:495. Any techniquefor producing monoclonal antibody can be employed, e.g., viral oroncogenic transformation of B lymphocytes. One animal system forpreparing hybridomas is the murine system. Hybridoma production in themouse is a very well-established procedure. Immunization protocols andtechniques for isolation of immunized splenocytes for fusion are knownin the art. Fusion partners (e.g., murine myeloma cells) and fusionprocedures are also known (see, e.g., Harlow and Lane (1988) Antibodies,A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor N.Y.).

The combination of antibodies is useful for enhancement of an immuneresponse against a hyperproliferative disease by blockade of O8E andPD-1 and/or CTLA-4. In a preferred embodiment, the antibodies of thepresent disclosure are human antibodies. For example, these moleculescan be administered to cells in culture, in vitro or ex vivo or to humansubjects, e.g., in vivo, to enhance immunity in a variety of situations.Accordingly, in one aspect, this disclosure provides a method ofmodifying an immune response in a subject comprising administering tothe subject an antibody combination or a combination of antigen-bindingportions thereof, of this disclosure such that the immune response inthe subject is modified. Preferably, the response is enhanced,stimulated or up-regulated. In another embodiment, the instantdisclosure provides a method of altering adverse events associated withtreatment of a hyperproliferative disease with an immunostimulatorytherapeutic agent, comprising administering an anti-O8E antibody and asubtherapeutic dose of anti-CTLA-4 or anti-PD-1 antibody to a subject.

Blockade of O8E, PD-1 and CTLA-4 by antibodies can enhance the immuneresponse to cancerous cells in the patient. Cancers whose growth may beinhibited using the antibodies of the instant disclosure include cancerstypically responsive to immunotherapy. Representative examples ofcancers for treatment with the combination therapy of the instantdisclosure include melanoma (e.g., metastatic malignant melanoma), renalcancer, prostate cancer, breast cancer, colon cancer and lung cancer.Examples of other cancers that may be treated using the methods of theinstant disclosure include bone cancer, pancreatic cancer, skin cancer,cancer of the head or neck, cutaneous or intraocular malignant melanoma,uterine cancer, ovarian cancer, rectal cancer, cancer of the analregion, stomach cancer, testicular cancer, uterine cancer, carcinoma ofthe fallopian tubes, carcinoma of the endometrium, carcinoma of thecervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin'sDisease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of thesmall intestine, cancer of the endocrine system, cancer of the thyroidgland, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,chronic or acute leukemias including acute myeloid leukemia, chronicmyeloid leukemia, acute lymphoblastic leukemia, chronic lymphocyticleukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of thebladder, cancer of the kidney or ureter, carcinoma of the renal pelvis,neoplasm of the central nervous system (CNS), primary CNS lymphoma,tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitaryadenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer,T-cell lymphoma, environmentally induced cancers including those inducedby asbestos and combinations of said cancers. The present disclosure isalso useful for treatment of metastatic cancers.

In certain embodiments, the combination of therapeutic antibodiesdiscussed herein may be administered concurrently as a singlecomposition in a pharmaceutically acceptable carrier or concurrently asseparate compositions with each antibody in a pharmaceuticallyacceptable carrier. In another embodiment, the combination oftherapeutic antibodies can be administered sequentially. For example, ananti-O8E antibody and an anti-PD-1 antibody can be administeredsequentially, such as anti-O8E being administered first and anti-PD-1second or anti-PD-1 being administered first and anti-O8E second.Furthermore, if more than one dose of the combination therapy isadministered sequentially, the order of the sequential administrationcan be reversed or kept in the same order at each time point ofadministration, sequential administrations may be combined withconcurrent administrations or any combination thereof. For example, thefirst administration of a combination anti-O8E antibody and anti-PD-1antibody may be concurrent, the second administration may be sequentialwith anti-O8E first and anti-PD-1 second and the third administrationmay be sequential with anti-PD-1 first and anti-O8E second, etc. Anotherrepresentative dosing scheme may involve a first administration that issequential with anti-PD-1 first and anti-O8E second and subsequentadministrations may be concurrent.

Optionally, the combination of anti-O8E and anti-CTLA-4 and/or anti-PD-1antibodies can be further combined with an immunogenic agent, such ascancerous cells, purified tumor antigens (including recombinantproteins, peptides and carbohydrate molecules), cells and cellstransfected with genes encoding immune stimulating cytokines (He et al.(2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccinesthat can be used include peptides of melanoma antigens, such as peptidesof gp100, MAGE antigens, Trp-2, MARTI and/or tyrosinase or tumor cellstransfected to express the cytokine GM-CSF (discussed further below).

A combined O8E and PD-1 and/or CTLA-4 blockade can be further combinedwith a vaccination protocol. Many experimental strategies forvaccination against tumors have been devised (see Rosenberg, S. (2000)Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62;Logothetis, C., 2000, ASCO Educational Book Spring: 300-302; Khayat, D.(2000) ASCO Educational Book Spring: 414-428; Foon, K. (2000) ASCOEducational Book Spring: 730-738; see also Restifo and Sznol, CancerVaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.), 1997, Cancer:Principles and Practice of Oncology. Fifth Edition). In one of thesestrategies, a vaccine is prepared using autologous or allogeneic tumorcells. These cellular vaccines have been shown to be most effective whenthe tumor cells are transduced to express GM-CSF. GM-CSF has been shownto be a potent activator of antigen presentation for tumor vaccination(Dranoff et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90: 3539-43).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg (1999) Immunity 10:281-7). In many cases, thesetumor specific antigens are differentiation antigens expressed in thetumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. In certain embodiments, a combined O8E andPD-1 and/or CTLA-4 blockade using the antibody compositions describedherein may be used in conjunction with a collection of recombinantproteins and/or peptides expressed in a tumor in order to generate animmune response to these proteins. These proteins are normally viewed bythe immune system as self-antigens and are, therefore, tolerant to them.The tumor antigen may also include the protein telomerase, which isrequired for the synthesis of telomeres of chromosomes and which isexpressed in more than 85% of human cancers and in only a limited numberof somatic tissues (Kim et al. (1994) Science 266: 2011-2013). (Thesesomatic tissues may be protected from immune attack by various means).Tumor antigen may also be “neo-antigens” expressed in cancer cellsbecause of somatic mutations that alter protein sequence or createfusion proteins between two unrelated sequences (i.e., bcr-abl in thePhiladelphia chromosome) or idiotype from B cell tumors.

Other tumor vaccines may include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which may be used in conjunction with O8Eblockade is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot & Srivastava(1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs mayalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As amethod of vaccination, DC immunization may be effectively furthercombined with a combined O8E and PD-1 and/or CTLA-4 blockade to activatemore potent anti-tumor responses.

A combined O8E and PD-1 and/or CTLA-4 blockade may also be furthercombined with standard cancer treatments. For example, a combined O8Eand PD-1 and/or CTLA-4 blockade may be effectively combined withchemotherapeutic regimes. In these instances, as is observed with thecombination of anti-O8E and anti-CTLA-4 and/or anti-PD-1 antibodies, itmay be possible to reduce the dose of other chemotherapeutic reagentadministered with the combination of the instant disclosure (Mokyr etal. (1998) Cancer Research 58: 5301-5304). The scientific rationalebehind the combined use of O8E and PD-1 and/or CTLA-4 blockade withchemotherapy is that cell death, which is a consequence of the cytotoxicaction of most chemotherapeutic compounds, should result in increasedlevels of tumor antigen in the antigen presentation pathway. Othercombination therapies that may result in synergy with a combined O8E andPD-1 and/or CTLA-4 blockade through cell death include radiation,surgery or hormone deprivation. Each of these protocols creates a sourceof tumor antigen in the host. Angiogenesis inhibitors may also becombined with a combined O8E and PD-1 and/or CTLA-4 blockade. Inhibitionof angiogenesis leads to tumor cell death, which may also be a source oftumor antigen to be fed into host antigen presentation pathways.

A combination of O8E and PD-1 and/or CTLA-4 blocking antibodies can alsobe used in combination with bispecific antibodies that target Fcα or Fcγreceptor-expressing effector cells to tumor cells (see, e.g., U.S. Pat.Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used totarget two separate antigens. For example anti-Fc receptor/anti tumorantigen (e.g., Her-2/neu) bispecific antibodies have been used to targetmacrophages to sites of tumor. This targeting may more effectivelyactivate tumor specific responses. The T cell arm of these responseswould by augmented by the use of a combined O8E and PD-1 and/or CTLA-4blockade. Alternatively, antigen may be delivered directly to DCs by theuse of bispecific antibodies which bind to tumor antigen and a dendriticcell specific cell surface marker.

In another example, a combination of anti-PD-1 and anti-CTLA-4antibodies can be used in conjunction with anti-neoplastic antibodies,such as Rituxan® (rituximab), Herceptin® (trastuzumab), Bexxar®(tositumomab), Zevalin® (ibritumomab), Campath® (alemtuzumab),Lymphocide® (eprtuzumab), Avastin® (bevacizumab) and Tarceva®(erlotinib) and the like. By way of example and not wishing to be boundby theory, treatment with an anti-cancer antibody or an anti-cancerantibody conjugated to a toxin can lead to cancer cell death (e.g.,tumor cells) which would potentiate an immune response mediated by O8E,CTLA-4 or PD-1. In an exemplary embodiment, a treatment of ahyperproliferative disease (e.g., a cancer tumor) may include ananti-cancer antibody in combination with anti-O8E and anti-PD-1 and/oranti-CTLA-4 antibodies, concurrently or sequentially or any combinationthereof, which may potentiate an anti-tumor immune responses by thehost.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation ofproteins, which are expressed by the tumors and which areimmunosuppressive. These include, among others, TGF-β (Kehrl, J. et al.(1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard, M. & O'Garra, A.(1992) Immunology Today 13: 198-200) and Fas ligand (Hahne, M. et al.(1996) Science 274: 1363-1365). In another example, antibodies to eachof these entities may be further combined with an anti-O8E and anti-PD-1and/or anti-CTLA-4 combination to counteract the effects ofimmunosuppressive agents and favor anti-tumor immune responses by thehost.

Other antibodies that may be used to activate host immune responsivenesscan be further used in combination with an anti-O8E and anti-PD-1 and/oranti-CTLA-4 combination. These include molecules on the surface ofdendritic cells that activate DC function and antigen presentation.Anti-CD40 antibodies are able to substitute effectively for T cellhelper activity (Ridge, J. et al. (1998) Nature 393: 474-478) and havebeen shown efficacious in conjunction with anti-CTLA-4 (Ito, N. et al.(2000) Immunobiology 201 (5) 527-40). Activating antibodies to T cellcostimulatory molecules, such as OX-40 (Weinberg, A. et al. (2000)Immunol 164: 2160-2169), 4-1BB (Melero, I. et al. (1997) Nature Medicine3: 682-685 (1997), PD-1 (del R10 et al. (2005) Eur J Immunol.35:3545-60) and ICOS (Hutloff, A. et al. (1999) Nature 397: 262-266) mayalso provide for increased levels of T cell activation.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses. A combined O8E and PD-1 and/or CTLA-4blockade can be used to increase the effectiveness of the donorengrafted tumor specific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to antigen-specific Tcells against tumor (Greenberg, R. & Riddell, S. (1999) Science 285:546-51). These methods may also be used to activate T cell responses toinfectious agents such as CMV. Ex vivo activation in the presence ofanti-O8E and anti-PD-1 and/or anti-CTLA-4 antibodies may be expected toincrease the frequency and activity of the adoptively transferred Tcells.

As set forth herein organs can exhibit immune-related adverse eventsfollowing immunostimulatory therapeutic antibody therapy, such as the GItract (diarrhea and colitis) and the skin (rash and pruritis) aftertreatment with anti-CTLA-4 antibody. For example, non-colonicgastrointestinal immune-related adverse events have also been observedin the esophagus (esophagitis), duodenum (duodenitis) and ileum(ileitis) after anti-CTLA-4 antibody treatment.

In certain embodiments, the present disclosure provides a method foraltering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering a anti-O8E antibody and a subtherapeutic dose ofanti-CTLA-4 antibody to a subject. For example, the methods of thepresent disclosure provide for a method of reducing the incidence ofimmunostimulatory therapeutic antibody-induced colitis or diarrhea byadministering a non-absorbable steroid to the patient. Because anypatient who will receive an immunostimulatory therapeutic antibody is atrisk for developing colitis or diarrhea induced by such an antibody,this entire patient population is suitable for therapy according to themethods of the present disclosure. Although steroids have beenadministered to treat inflammatory bowel disease (IBD) and preventexacerbations of IBD, they have not been used to prevent (decrease theincidence of) IBD in patients who have not been diagnosed with IBD. Thesignificant side effects associated with steroids, even non-absorbablesteroids, have discouraged prophylactic use.

In further embodiments, a combination O8E and PD-1 and/or CTLA-4blockade (i.e., immunostimulatory therapeutic antibodies anti-O8E andanti-PD-1 and/or anti-CTLA-4) can be further combined with the use ofany non-absorbable steroid. As used herein, a “non-absorbable steroid”is a glucocorticoid that exhibits extensive first pass metabolism suchthat, following metabolism in the liver, the bioavailability of thesteroid is low, i.e., less than about 20%. In one embodiment of thisdisclosure, the non-absorbable steroid is budesonide. Budesonide is alocally-acting glucocorticosteroid, which is extensively metabolized,primarily by the liver, following oral administration. ENTOCORT EC®(Astra-Zeneca) is a pH- and time-dependent oral formulation ofbudesonide developed to optimize drug delivery to the ileum andthroughout the colon. ENTOCORT EC is approved in. the U.S. for thetreatment of mild to moderate Crohn's disease involving the ileum and/orascending colon. The usual oral dosage of ENTOCORT EC® for the treatmentof Crohn's disease is 6 to 9 mg/day. ENTOCORT EC® is released in theintestines before being absorbed and retained in the gut mucosa. Once itpasses through the gut mucosa target tissue, ENTOCORT EC® is extensivelymetabolized by the cytochrome P450 system in the liver to metaboliteswith negligible glucocorticoid activity. Therefore, the bioavailabilityis low (about 10%). The low bioavailability of budesonide results in animproved therapeutic ratio compared to other glucocorticoids with lessextensive first-pass metabolism. Budesonide results in fewer adverseeffects, including less hypothalamic-pituitary suppression, thansystemically-acting corticosteroids. However, chronic administration ofENTOCORT EC® can result in systemic glucocorticoid effects such ashypercorticism and adrenal suppression. See PDR 58^(th) ed. 2004;608-610.

In still further embodiments, a combination 0 8E and PD-1 and/or CTLA-4blockade (i.e., immunostimulatory therapeutic antibodies anti-O8E andanti-PD-1 and/or anti-CTLA-4) in conjunction with a non-absorbablesteroid can be further combined with a salicylate. Salicylates include5-ASA agents such as, for example: sulfasalazine (AZULFIDINE®, Pharmacia& UpJohn); olsalazine (DIPENTUM®, Pharmacia & UpJohn); balsalazide(COLAZAL®, Salix Pharmaceuticals, Inc.); and mesalamine (ASACOL®,Procter & Gamble Pharmaceuticals; PENTASA®, Shire US; CANASA®, AxcanScandipharm, Inc.; ROWASA®, Solvay).

In accordance with the methods of the present disclosure, a salicylateadministered in combination with anti-O8E and anti-PD-1 and/oranti-CTLA-4 antibodies and a non-absorbable steroid can includes anyoverlapping or sequential administration of the salicylate and thenon-absorbable steroid for the purpose of decreasing the incidence ofcolitis induced by the immunostimulatory antibodies. Thus, for example,methods for reducing the incidence of colitis induced by theimmunostimulatory antibodies according to the present disclosureencompass administering a salicylate and a non-absorbable concurrentlyor sequentially (e.g., a salicylate is administered 6 hours after anon-absorbable steroid) or any combination thereof. Further, accordingto the present disclosure, a salicylate and a non-absorbable steroid canbe administered by the same route (e.g., both are administered orally)or by different routes (e.g., a salicylate is administered orally and anon-absorbable steroid is administered rectally), which may differ fromthe route(s) used to administer the anti-O8E, anti-PD-1 and anti-CTLA-4antibodies.

The compositions (e.g., human antibodies, multispecific and bispecificmolecules and immunoconjugates) of this disclosure which have complementbinding sites, such as portions from IgG1, -2 or -3 or IgM which bindcomplement, can also be used in the presence of complement. In oneembodiment, ex vivo treatment of a population of cells comprising targetcells with a binding agent of this disclosure and appropriate effectorcells can be supplemented by the addition of complement or serumcontaining complement. Phagocytosis of target cells coated with abinding agent of this disclosure can be improved by binding ofcomplement proteins. In another embodiment target cells coated with thecompositions (e.g., human antibodies, multispecific and bispecificmolecules) of this disclosure can also be lysed by complement. In yetanother embodiment, the compositions of this disclosure do not activatecomplement.

The compositions (e.g., human antibodies, multispecific and bispecificmolecules and immunoconjugates) of this disclosure can also beadministered together with complement. Accordingly, within the scope ofthis disclosure are compositions comprising human antibodies,multispecific or bispecific molecules and serum or complement. Thesecompositions are advantageous in that the complement is located in closeproximity to the human antibodies, multispecific or bispecificmolecules. Alternatively, the human antibodies, multispecific orbispecific molecules of this disclosure and the complement or serum canbe administered separately.

Accordingly, patients treated with antibody compositions of thisdisclosure can be additionally administered (prior to, simultaneouslywith or following administration of a human antibody of this disclosure)with another therapeutic agent, such as a cytotoxic or radiotoxic agent,which enhances or augments the therapeutic effect of the humanantibodies.

In other embodiments, the subject can be additionally treated with anagent that modulates, e.g., enhances or inhibits, the expression oractivity of Fcγ or Fcγ receptors by, for example, treating the subjectwith a cytokine. Preferred cytokines for administration during treatmentwith the multispecific molecule include of granulocytecolony-stimulating factor (G-CSF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), interferon-γ (IFN-γ) and tumornecrosis factor (TNF).

The compositions (e.g., human antibodies, multispecific and bispecificmolecules) of this disclosure can also be used to target cellsexpressing FcγR or O8E, for example for labeling such cells. For suchuse, the binding agent can be linked to a molecule that can be detected.Thus, this disclosure provides methods for localizing ex vivo or invitro cells expressing Fc receptors, such as FcγR or O8E. The detectablelabel can be, e.g., a radioisotope, a fluorescent compound, an enzyme oran enzyme co-factor.

In a particular embodiment, this disclosure provides methods fordetecting the presence of O8E antigen in a sample or measuring theamount of O8E antigen, comprising contacting the sample and a controlsample, with a human monoclonal antibody or an antigen binding portionthereof, which specifically binds to O8E, under conditions that allowfor formation of a complex between the antibody or portion thereof andO8E. The formation of a complex is then detected, wherein a differencecomplex formation between the sample compared to the control sample isindicative the presence of O8E antigen in the sample.

In other embodiments, this disclosure provides methods for treating aO8E mediated disorder in a subject.

In yet another embodiment, immunoconjugates of this disclosure can beused to target compounds (e.g., therapeutic agents, labels, cytotoxins,radiotoxins immunosuppressants, etc.) to cells which have O8E cellsurface receptors by linking such compounds to the antibody. Forexample, an anti-O8E antibody can be conjugated to UPT, as described inU.S. patent application Ser. Nos. 10/160,972, 10/161,233, 10/161,234,11/134,826, 11/134,685 and U.S. Provisional Patent Application No.60/720,499 and/or any of the toxin compounds described in U.S. Pat. Nos.6,281,354 and 6,548,530, US patent publication Nos. 20030050331,20030064984, 20030073852 and 20040087497 or published in WO 03/022806,which are hereby incorporated by reference in their entireties. Thus,this disclosure also provides methods for localizing ex vivo or in vivocells expressing O8E (e.g., with a detectable label, such as aradioisotope, a fluorescent compound, an enzyme or an enzyme co-factor).Alternatively, the immunoconjugates can be used to kill cells which haveO8E cell surface receptors by targeting cytotoxins or radiotoxins toO8E.

The present disclosure is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

EXAMPLES Example 1 Generation of Human Monoclonal Antibodies Against O8E

This Example discloses the generation of human monoclonal antibodiesthat specifically bind to human O8E (a/k/a B7H4, B7S1 and B7x).

Antigen

CHO and HEK-293 cells were transfected with O8E using standardrecombinant transfection methods and used as antigen for immunization.In addition, recombinant O8E alone was also used as antigen forimmunization,

Transgenic HuMAb Mouse® and KM Mouse®

Fully human monoclonal antibodies to O8E were prepared using the HCo7and HCo12 strains of the transgenic HuMAb Mouse® and the KM strain oftransgenic transchromosomic mice, each of which express human antibodygenes. In each of these mouse strains, the endogenous mouse kappa lightchain gene has been homozygously disrupted as described in Chen et al.(1993) EMBO J. 12:811-820 and the endogenous mouse heavy chain gene hasbeen homozygously disrupted as described in Example 1 of PCT PublicationWO 01/09187. Each of these mouse strains carries a human kappa lightchain transgene, KCo5, as described in Fishwild et al. (1996) NatureBiotechnology 14:845-851. The HCo7 strain carries the HCo7 human heavychain transgene as described in U.S. Pat. Nos. 5,545,806; 5,625,825; and5,545,807. The HCo12 strain carries the HCo12 human heavy chaintransgene as described in Example 2 of PCT Publication WO 01/09187. TheKM Mouse® strain contains the SC20 transchromosome as described in PCTPublication WO 02/43478.

HuMAb and KM Immunizations:

To generate fully human monoclonal antibodies to O8E, mice of the HuMAbMouse® and KM Mouse® were immunized with CHO-O8E transfected cells,HEK293-O8E transfected cells and/or purified recombinant O8E protein.General immunization schemes for HuMAb Mouse® are described in Lonberg,N. et al (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996)Nature Biotechnology 14: 845-851 and PCT Publication WO 98/24884. Themice were 6-16 weeks of age upon the first infusion of antigen. Apurified recombinant preparation (5-50 μg) of O8E protein was used toimmunize the HuMAb Mice™ and KM Mice™.

Transgenic mice were immunized twice with antigen in complete Freund'sadjuvant adjuvant either intraperitonealy (IP) or subcutaneously (Sc),followed by 3-21 days IP or SC immunization (up to a total of 11immunizations) with the antigen in incomplete Freund's adjuvant. Theimmune response was monitored by retroorbital bleeds. The plasma wasscreened by ELISA (as described below) and mice with sufficient titersof anti-O8E human immunogolobulin were used for fusions. Mice wereboosted intravenously with antigen 3 and 2 days before sacrifice andremoval of the spleen. Typically, 10-35 fusions for each antigen wereperformed. Several dozen mice were immunized for each antigen.

Selection of HuMb Mice™ or KM Mice™ Producing Anti-O8E Antibodies:

To select HuMab Mice™ or KM Mice™ producing antibodies that bound O8Esera from immunized mice was tested by ELISA as described by Fishwild,D. et al. (1996) (supra). Briefly, microtiter plates were coated withpurified recombinant O8E at 1-2 μg/ml in PBS, 50 μl/wells incubated 4°C. overnight then blocked with 2001/well of 5% chicken serum inPBS/Tween (0.05%). Dilutions of plasma from O8E-immunized mice wereadded to each well and incubated for 1-2 hours at ambient temperature.The plates were washed with PBS/Tween and then incubated with agoat-anti-human IgG Fc polyclonal antibody conjugated with horseradishperoxidase (HRP) for 1 hour at room temperature. After washing, theplates were developed with ABTS substrate (Sigma, A-1888, 0.22 mg/ml)and analyzed by spectrophotometer at OD 415-495. Mice that developed thehighest titers of anti-O8E antibodies were used for fusions. Fusionswere performed as described below and hybridoma supernatants were testedfor anti-O8E activity by ELISA and FACS.

Generation of Hybridomas Producing Human Monoclonal Antibodies to O8E:

The mouse splenocytes, isolated from the HuMab Mice™ and KM Mice™, werefused with PEG to a mouse myeloma cell line either using PEG based uponstandard protocols. The resulting hybridomas were then screened for theproduction of antigen-specific antibodies. Single cell suspensions ofsplenic lymphocytes from immunized mice were fused to one-fourth thenumber of SP2/0 nonsecreting mouse myeloma cells (ATCC, CRL 1581) with50% PEG (Sigma). Cells were plated at approximately 1×10⁵ cells/well inflat bottom microtiter plate, followed by a about two week incubation inselective medium containing 10% fetal bovine serum (Hyclone, Logan,Utah), 10% P388DI (ATCC, CRL TIB-63) conditioned medium, 3-5% origen(IGEN) in DMEM (Mediatech, CRL 10013, with high glucose, L-glutamine andsodium pyruvate) plus 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 mg/mlgentamycin and 1×HAT (Sigma, CRL P-7185). After one to two weeks, cellswere cultured in medium in which HAT was replaced with HT. Individualwells were then screened by ELISA and FACS (described above) for humananti-O8E monoclonal IgG antibodies. The positive clones were thenscreened for O8E positive antibodies on O8E recombinant protein by ELISAor on O8E expressing cells, for example CHO-O8E transfected cells, byFACS. Briefly, O8E-expressing cells were freshly harvested from tissueculture flasks and a single cell suspension prepared. O8E-expressingcell suspensions were either stained with primary antibody directly orafter fixation with 1% paraformaldehyde in PBS. Approximately onemillion cells were resuspended in PBS containing 0.5% BSA and 50-200μg/ml of primary antibody and incubated on ice for 30 minutes. The cellswere washed twice with PBS containing 0.1% BSA, 0.01% NaN₃, resuspendedin 100 μl of 1:100 diluted FITC-conjugated goat-anti-human IgG (JacksonImmunoResearch, West Grove, Pa.) and incubated on ice for an additional30 minutes. The cells were again washed twice, resuspended in 0.5 ml ofwash buffer and analyzed for fluorescent staining on a FACSCaliburcytometer (Becton-Dickinson, San Jose, Calif.).

Once extensive hybridoma growth occurred, medium was monitored usuallyafter 10-14 days. The antibody-secreting hybridomas were replated,screened again and, if still positive for human IgG, anti-O8E monoclonalantibodies were subcloned at least twice by limiting dilution. Thestable subclones were then cultured in vitro to generate small amountsof antibody in tissue culture medium for further characterization.

Hybridoma clones 1G11, 2A7, 2F9, 12E1 and 13D12 were selected forfurther analysis.

Example 2 Structural Characterization of Human Monoclonal Antibodies1G11, 2A7, 2F9, 12E1 and 13D12

This Example discloses sequence analysis five (5) human monoclonalantibodies that specifically bind to O8E.

The cDNA sequences encoding the heavy and light chain variable regionsof the 1G11, 2A7, 2F9, 12E1 and 13D12 monoclonal antibodies wereobtained from the 1G11, 2A7, 2F9, 12E1 and 13D12 hybridomas,respectively, using standard PCR techniques and were sequenced usingstandard DNA sequencing techniques.

The nucleotide and amino acid sequences of the heavy chain variableregion of 1G11 are shown in FIG. 1A and in SEQ ID NOs: 41 and 1,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 1G11 are shown in FIG. 1B and in SEQ ID NO: 46 and 6,respectively.

Comparison of the 1G1 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 1G11 heavy chain utilizes a VH segment from human germline V_(H)4-34. The alignment of the 1G11 VH sequence to the germline VH 4-34sequence is shown in FIG. 6. Further analysis of the 1G11 VH sequenceusing the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 1A and 6 and in SEQ ID NOs: 11, 16 and 21, respectively.

Comparison of the 1G11 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 10G1 light chain utilizes a V_(L) segment from human germline V_(K)A27. The alignment of the 1G11 V_(L) sequence to the germline V_(K) A27sequence is shown in FIG. 9. Further analysis of the 1G11 V_(L) sequenceusing the Kabat system of CDR region determination led to thedelineation of the light chain CDR1, CDR2 and CD3 regions as shown inFIGS. 1B and 9 and in SEQ ID NOs: 26, 31 and 36, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 2A7 are shown in FIG. 2A and in SEQ ID NO: 42 and 2,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 2A7 are shown in FIG. 2B and in SEQ ID NO: 47 and 7,respectively.

Comparison of the 2A7 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 2A7 heavy chain utilizes a V_(H) segment from human germline V_(H)3-53 and a JH segment from human germline JH 6b. The alignment of the2A7 V_(H) sequence to the germline V_(H) 3-53 sequence is shown in FIG.7. Further analysis of the 2A7 V_(H) sequence using the Kabat system ofCDR region determination led to the delineation of the heavy chain CDR1,CDR2 and CD3 regions as shown in FIGS. 2A and 7 and in SEQ ID NOs: 12,17 and 22, respectively.

Comparison of the 2A7 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 2A7 light chain utilizes a VL segment from human germline VK A27.The alignment of the 2A7 VL sequence to the germline VK A27 sequence isshown in FIG. 9. Further analysis of the 2A7 V_(L) sequence using theKabat system of CDR region determination led to the delineation of thelight chain CDR1, CDR2 and CD3 regions as shown in FIGS. 2B and 9 and inSEQ ID NOs: 27, 32 and 37, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 2F9 are shown in FIG. 3A and in SEQ ID NO: 43 and 3,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 2F9 are shown in FIG. 3B and in SEQ ID NO: 48 and 8,respectively.

Comparison of the 2F9 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 2F9 heavy chain utilizes a VH segment from human germline VH 3-53and a JH segment from human germline JH 6b. The alignment of the 2F9 VHsequence to the germline VH 3-53 sequence is shown in FIG. 7. Furtheranalysis of the 2F9 VH sequence using the Kabat system of CDR regiondetermination led to the delineation of the heavy chain CDR1, CDR2 andCD3 regions as shown in FIGS. 3A and 7 and in SEQ ID NOs: 13, 18 and 23,respectively.

Comparison of the 2F9 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 2F9 light chain utilizes a VL segment from human germline VK A27.The alignment of the 2F9 VL sequence to the germline VK 27 sequence isshown in FIG. 9. Further analysis of the 2F9 VL sequence using the Kabatsystem of CDR region determination led to the delineation of the lightchain CDR1, CDR2 and CD3 regions as shown in FIGS. 3B and 9 and in SEQID NOs: 28, 33 and 38, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 12E1 are shown in FIG. 4A and in SEQ ID NO: 44 and 4,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 12E1 are shown in FIG. 4B and in SEQ ID NO: 49 and 9,respectively.

Comparison of the 12E1 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 12E1 heavy chain utilizes a VH segment from human germline VH 3-9, aD segment from human germline 3-10 and a JH segment from human germlineJH 6b. The alignment of the 12E1 VH sequence to the germline V_(H) 3-9sequence is shown in FIG. 8. Further analysis of the 12E1 VH sequenceusing the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 3A and 8 and in SEQ ID NOs: 14, 19 and 24, respectively.

Comparison of the 12E1 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 12E1 light chain utilizes a VL segment from human germline V_(K) L6and a JK segment from human germline JK 1. The alignment of the 12E1 VLsequence to the germline VK L6 sequence is shown in FIG. 10. Furtheranalysis of the 12E1 VL sequence using the Kabat system of CDR regiondetermination led to the delineation of the light chain CDR1, CDR2 andCD3 regions as shown in FIGS. 3B and 10 and in SEQ ID NOs: 29, 34 and39, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 13D12 are shown in FIG. 5A and in SEQ ID NO: 45 and 5,respectively.

The nucleotide and amino acid sequences of the light chain variableregion of 13D12 are shown in FIG. 5B and in SEQ ID NO: 50 and 10,respectively.

Comparison of the 13D12 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 13D12 heavy chain utilizes a V_(H) segment from human germline V_(H)4-34. The alignment of the 13D12 VH sequence to the germline V_(H) 4-34sequence is shown in FIG. 6. Further analysis of the 13D12 VH sequenceusing the Kabat system of CDR region determination led to thedelineation of the heavy chain CDR1, CDR2 and CD3 regions as shown inFIGS. 5A and 6 and in SEQ ID NOs: 15, 20 and 25, respectively.

Comparison of the 13D12 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 13D12 light chain utilizes a VL segment from human germline VK A27.The alignment of the 13D12 VL sequence to the germline VK A27 sequenceis shown in FIG. 9. Further analysis of the 13D12 VL sequence using theKabat system of CDR region determination led to the delineation of thelight chain CDR1, CDR2 and CD3 regions as shown in FIGS. 5B and 9 and inSEQ ID NOs: 30, 35 and 40, respectively.

Example 3 Characterization of Binding Specificity of Anti-O8E HumanMonoclonal Antibodies

This Example discloses a comparison of anti-O8E antibodies on binding toimmunopurified O8E performed by standard ELISA to examine thespecificity of binding for O8E.

Recombinant His-tagged and myc-tagged O8E was coated on a plateovernight, then tested for binding against the anti-O8E human monoclonalantibodies 2A7, 12E1 and 13D12. Standard ELISA procedures wereperformed. The anti-O8E human monoclonal antibodies were added at aconcentration of 1 μg/ml and titrated down at 1:2 serial dilutions.Goat-anti-human IgG (Fc or kappa chain-specific) polyclonal antibodyconjugated with horseradish peroxidase (HRP) was used as secondaryantibody.

Recombinant B7H4-Ig was purified from supernatants of 293T cellstransfected with a B7H4-Ig construct by chromatography using protein A.An ELISA plate was coated with the human antibodies, followed byaddition of purified protein and then detection with the rabbitanti-B7H4 antisera. See, FIG. 11A. Recombinant Penta-B7H4 protein with aC-9 tag was purified from supernatants of 293T cells transfected with aPenta-B7H4-C9 construct by chromatography using a 2A7 affinity column.An ELISA plate was coated with anti-mouse Fc, followed by monoclonalanti-C9 (0.6 ug/ml), then titrated Penta-B7H4 as indicated, then thehuman antibodies at 1 ug/ml. Coated anti-mouse Fc followed by M-anti-C9(0.6 ug/ml), then titrated Penta-O8E as indicated, then humabs @ 1ug/ml. See, FIG. 11B.

The anti-O8E human monoclonal antibodies 2A7, 12E1 and 13D12 bound withhigh specificity to O8E.

Example 4 Characterization of Anti-O8E Antibody Binding to O8E Expressedon the Surface of Breast Cancer Carcinoma Cell Lines

This Example discloses the testing of anti-O8E antibodies for binding toCHO-O8E (a/k/a B7H4, B7S1 and B7x) transfectants and breast cellcarcinoma cells expressing O8E on their cell surface by flow cytometry.

A CHO cell line transfected with O8E as well as the breast cellcarcinoma cell line SKBR3 (ATCC Accession No. HTB-30) were tested forantibody binding. Binding of the HuMAb 2A7 anti-O8E human monoclonalantibody was assessed by incubating 1×10⁵ cells with 2A7 at aconcentration of 1 μg/ml. The cells were washed and binding was detectedwith a FITC-labeled anti-human IgG Ab. Flow cytometric analyses wereperformed using a FACScan flow cytometry (Becton Dickinson, San Jose,Calif.). The results are shown in FIGS. 12 and 13.

These data demonstrate that the anti-O8E HuMAbs bind to O8E expressingCHO cells and to an exemplary breast cell carcinoma cell line.

Example 5 Scatchard Analysis of Binding Affinity of Anti-O8E MonoclonalAntibodies

This Example discloses the testing of human monoclonal antibodies 1G11,2F9, 2A7, 12E1 and 13D12 monoclonal antibodies for binding affinity to aO8E transfected HEK cell line using a Scatchard analysis.

HEK cells were transfected with fill length O8E using standardtechniques and grown in RPMI media containing 10% fetal bovine serum(FBS). (FIG. 12 presents FACs analysis of these HEK-O8E cells with the2A7 human anti-O8E monoclonal antibody.) The cells were trypsinized andwashed once in Tris based binding buffer (24 mM Tris pH 7.2, 137 mMNaCl, 2.7 mM KCl, 2 mM Glucose, 1 mM CaCl₂, 1 mM MgCl₂, 0.1% BSA) andthe cells were adjusted to 2×10⁶ cells/ml in binding buffer. Milliporeplates (MAFB NOB) were coated with 1% nonfat dry milk in water andstored a 4° C. overnight. The plates were washed three times with 0.2 mlof binding buffer. Fifty microliters of buffer alone was added to themaximum binding wells (total binding). Twenty-five microliters of bufferalone was added to the control wells (non-specific binding). Varyingconcentration of ¹²⁵I-anti-O8E antibody was added to all wells in avolume of 25 μl. (In some cases FITC labeled antibodies were used forthe titration since unlabeled material was not available, binding may becompromised in these instances.) Varying concentrations of unlabeledantibody at 100 fold excess was added in a volume of 25 μl to controlwells and 25 μl of O8E transfected CHO cells (2×10⁶ cells/ml) in bindingbuffer were added to all wells. The plates were incubated for 2 hours at200 RPM on a shaker at 4° C. At the completion of the incubation theMillipore plates were washed three times with 0.2 ml of cold wash buffer(24 mM Tris pH 7.2, 500 mM NaCl, 2.7 mM KCl, 2 mM Glucose, 1 mM CaCl₂, 1mM MgCl₂, 0.1% BSA.). The filters were removed and counted in a gammacounter. Evaluation of equilibrium binding was performed using singlesite binding parameters with the Prism software (San Diego, Calif.).

Data were analyzed by non-linear regression using a sigmoidal doseresponse (PRIZM™) and resulted in calculation of an EC50, which was usedto rank the antibodies as illustrated in Table 2. The EC50 valuescalculated in these experiments are qualitative measures of antibodyaffinity and do not represent absolute affinities for O8E.

TABLE 2 Antibody EC50 95% CI 2F9.E6-FITC 407 pM 250 to 663 pM 13D12.G10746 pM 569 to 979 pM 2A7.C11 750 pM 519 pM to 1 nM 1G11.H11-FITC 1.69 nM1.4 to 2.0 nM 12E1.G9* 19.8 pM 14 to 27.6 nM *BOTTOM and TOP valuesadjusted as constants to compensate for incomplete curve.

Example 6 Internalization of Anti-O8E Monoclonal Antibody

This Example demonstrates the testing of anti-O8E HuMAbs for the abilityto internalize into O8E-expressing CHO and breast carcinoma cells usinga Hum-Zap internalization assay. The Hum-Zap assay tests forinternalization of a primary human antibody through binding of asecondary antibody with affinity for human IgG conjugated to the toxinsaporin.

The O8E-expressing breast carcinoma cancer cell line SKBR3 was seeded at1.25×10⁴ cells/well in 100 μl wells overnight. The anti-O8E HuMAbantibodies 1G11, 2F9, 2A7, 12E1 or 13D12 were added to the wells at aconcentration of 10 pM. An isotype control antibody that is non-specificfor O8E was used as a negative control. The Hum-Zap (Advanced TargetingSystems, San Diego, Calif., IT-22-25) was added at a concentration of 11nM and plates were allowed to incubate for 72 hours. The plates werethen pulsed with 1.0 μCi of ³H-thymidine for 24 hours, harvested andread in a Top Count Scintillation Counter (Packard Instruments, Meriden,Conn.). The results are presented below in Table 3 and in FIGS. 14-15.The anti-O8E antibodies 1G11, 2F9, 2A7, 12E1 and 13D12 showed anantibody concentration dependent decrease in ³H-thymidine incorporationin O8E-expressing SKBR3 breast carcinoma cancer cells.

These data demonstrate that the anti-O8E antibodies 1G11, 2F9, 2A7, 12E1and 13D12 internalize into a breast carcinoma cancer cell line.

TABLE 3 Assay No. 1 Assay No. 2 Assay No. 3 % % % internalizationinternalization internalization Anti-O8E mean sd mean sd mean sd 2A7/C1129 12 17.5 3.5 40.7 2.7 2F9.E6 37 17 NT NT NT NT 1G11.H1 18  8 NT NT NTNT 13D12.G10 NT NT 12.1 2.5 12.2 2.8 12E1.G9 NT NT 10.4 18.5   4.3 2.7

The ranking for internalization efficiency was averaged over threeexperiments in SKBR3 and two experiments in CHO-O8E. The internalizationrankings, along with EC50s for binding to CHO-O8E, are presented inTables 4 and 5. Results show that internalization efficiency does notdirectly correlate with binding affinity, which suggests thatinternalization is epitope dependant.

TABLE 4 Internalization Efficiency Sorted by Internalization in theSBKR3 Breast Carcinoma Cell Line Internalization EC50 CHO- Anti-O8ESKBR3 CHO-O8E O8E binding 2F9.E6 1 3 407 pM 2A7.C11 2 1 750 pM 1G11.H1 34 1.69 nM 13D12.G10 4 2 746 pM 12E1.G9 5 5 19.8 pM

TABLE 5 Internalization Efficiency Sorted by Internalization in theCHO-O8E Cell Line Internalization EC50 CHO- Anti-O8E SKBR3 CHO-O8E O8Ebinding 2A7.C11 2 1 750 pM 13D12.G10 4 2 746 pM 2F9.E6 1 3 407 pM1G11.H1 3 4 1.69 nM 12E1.G9 5 5 19.8 pM

The internalization activity of the saporin conjugates in CHO-O8E wasmeasured with a dose response through a ˜500 pM to 1 pM range usinghuman monoclonal antibodies 2A7, 2F9 and 1G11. As illustrated in FIG.14, internalization was very efficient with EC50s in the low pM range. ACHO parental cell line and Hu IgG-SAP were used as negative controls andshowed no significant background toxicity or non-specificinternalization. Direct anti-O8E conjugates to SAP were used with SKBR3cells. The percentage of internalization (vs control) as a function of1g-SAP dose is presented in FIG. 15.

Example 7 Assessment of Cell Killing of a Toxin-Conjugated Anti-O8EAntibody on Breast Cell Carcinoma Cell Lines

This Example discloses the testing of anti-O8E monoclonal antibodiesconjugated to a toxin for the ability to kill an O8E⁺ breast cellcarcinoma cell line in a cell proliferation assay.

The anti-O8E HuMAb antibodies 1G11, 2F9, 2A7, 12E1 or 13D12 may beconjugated to a toxin via a linker, such as a peptidyl, hydrazone ordisulfide linker. An O8E-expressing breast carcinoma cancer cell line,such as SKBR3, is seeded at between about 1 and 3×10⁴ cells/wells in 100μl wells for 3 hours. An anti-O8E antibody-toxin conjugate is added tothe wells at a starting concentration of 30 nM and titrated down at 1:3serial dilutions. An isotype control antibody that is non-specific forO8E is used as a negative control. Plates are allowed to incubate for 69hours. The plates are then pulsed with 1.0 μCi of ³H-thymidine for 24hours, harvested and read in a Top Count Scintillation Counter (PackardInstruments, Meriden, Conn.). Anti-O8E antibodies are expected to showan antibody-toxin concentration dependent decrease in ³H-thymidineincorporation in O8E-expressing breast carcinoma cancer cells. This datademonstrates that the anti-O8E antibodies 1G11, 2F9, 2A7, 12E1 and 13D12are potentially cytotoxic to breast carcinoma cancer cells whenconjugated to a toxin.

Example 8 Assessment of ADCC Activity of Anti-O8E Antibody

This Example discloses the testing of anti-O8E monoclonal antibodies forthe ability to kill O8E⁺ cell lines in the presence of effector cellsvia antibody dependent cellular cytotoxicity (ADCC) in a fluorescencecytotoxicity assay.

Human effector cells were prepared from whole blood as follows. Humanperipheral blood mononuclear cells were purified from heparinized wholeblood by standard Ficoll-paque separation. The cells were resuspended inRPMI1640 media containing 10% FBS and 200 U/ml of human IL-2 andincubated overnight at 37° C. The following day, the cells werecollected and washed four times in culture media and resuspended at2×10⁷ cells/ml. Target O8E+ cells were incubated with BATDA reagent(Perkin Elmer, Wellesley, Mass.) at 2.5 μl BATDA per 1×106 targetcells/mL for 20 minutes at 37° C. The target cells were washed fourtimes, spun down and brought to a final volume of 1×10⁵ cells/ml.

The O8E⁺ cell line SKBR3 as well as an O8E transfected SKOV3 cell-linewere tested for antibody specific ADCC to the human anti-O8E monoclonalantibodies using the Delfia fluorescence emission analysis as follows.Each target cell line (100 μl of labeled target cells) was incubatedwith 50 μl of effector cells and 50 μl it of antibody. A target toeffector ratio of 1:50 was used throughout the experiments. In allstudies, a human IgG1 isotype control was used as a negative control.Following a 2000 rpm pulse spin and one hour incubation at 37° C., thesupernatants were collected, quick spun again and 20 μl of supernatantwas transferred to a flat bottom plate, to which 180 μl of Eu solution(Perkin Elmer, Wellesley, Mass.) was added and read in a RubyStar reader(BMG Labtech). The % lysis was calculated as follows: (samplerelease−spontaneous release*100)/(maximum release−spontaneous release),where the spontaneous release is the fluorescence from wells which onlycontain target cells and maximum release is the fluorescence from wellscontaining target cells and have been treated with 2% Triton-X. Cellcytotoxicity % lysis for the SKBR3 cells with anti-O8E antibodies 1G11,2F9 and 2A7 are presented in FIG. 17; cell cytotoxicity % lysis for theSKOV3-O8E transfected cell line with anti-O8E antibodies 1G11, 2F9 and2A7 are presented in FIG. 18; and concentration-dependent cellcytotoxicity % lysis for the SKBR3 cells with anti-O8E antibodies 2F9and 2A7 are presented in FIG. 19. Both of the O8E⁺-expressing cell linesSKBR3 and SKOV3-O8E showed antibody mediated cytotoxicity with the HuMAbanti-O8E antibodies 1G11, 2F9 and 2A7. These data demonstrate that HuMAbanti-O8E antibodies show specific cytotoxicity to O8E⁺ expressing cells.

Example 9 Treatment of In Vivo Tumor Xenograft Model Using Naked andCytotoxin-Conjugated Anti-O8E Antibodies

This Example discloses the in vivo treatment of mice implanted with abreast cell carcinoma tumor with toxin-conjugated anti-O8E antibodies toexamine the in vivo effect of the antibodies on tumor growth.

SKBR3 or other suitable breast cell carcinoma cells are expanded invitro using standard laboratory procedures. Male Ncr athymic nude mice(Taconic, Hudson, N.Y.) between 6-8 weeks of age are implantedsubcutaneously in the right flank with 7.5×10⁶ ACHN or A-498 cells in0.2 ml of PBS/Matrigel (1:1) per mouse. Mice are weighed and measuredfor tumors three dimensionally using an electronic caliper twice weeklyafter implantation. Tumor volumes are calculated as height×width×length.Mice with ACHN tumors averaging 270 mm³ or A498 tumors averaging 110 mm³are randomized into treatment groups. The mice are dosedintraperitoneally with PBS vehicle, toxin-conjugated isotype controlantibody or toxin-conjugated anti-O8E HuMAb on Day 0. Examples of toxincompounds that may be conjugated to the antibodies of the currentdisclosure are described in pending U.S. Patent Application designatedMEDX-0034US4. The mice receiving anti-O8E HuMAb are tested with threedifferent toxin compounds. Mice are monitored for tumor growth for 60days post dosing. Mice are euthanized when the tumors reached tumor endpoint (2000 mm³). Suitable anti-O8E antibodies conjugated to a toxinextend the mean time to reaching the tumor end point volume (2000 mm³)and slow tumor growth progression. Thus, treatment with such an anti-O8Eantibody-toxin conjugate has a direct in vivo inhibitory effect on tumorgrowth.

Example 10 Immunohistochemistry with Anti-O8E HuMAb 2A 7

This Example discloses that the anti-O8E HuMAb 2A7 to recognize O8E byimmunohistochemistry using normal mouse tissue arrays (IMGENEXHisto-Array; Imgenex Corp., San Diego, Calif.).

For immunohistochemistry, 2,000 μm tissue cores were used. After dryingfor 30 minutes, sections were fixed with acetone (at room temperaturefor 10 minutes) and air-dried for 5 minutes. Slides were rinsed in PBSand then pre-incubated with 10% normal goat serum in PBS for 20 min andsubsequently incubated with 10 μg/ml fitcylated 2A7 in PBS with 10%normal goat serum for 30 min at room temperature. Next, slides werewashed three times with PBS and incubated for 30 min with mouseanti-FITC (10 μg/ml DAKO) at room temperature. Slides were washed againwith PBS and incubated with Goat anti-mouse HRP conjugate (DAKO) for 30minutes at room temperature. Slides were washed again 3× with PBS.Diaminobenzidine (Sigma) was used as substrate, resulting in brownstaining. After washing with distilled water, slides werecounter-stained with hematoxyllin for 1 min. Subsequently, slides werewashed for 10 secs in running distilled water and mounted in glycergel(DAKO). The results of these studies are presented in Table 6.

TABLE 6 Immunoreactivity of O8E in Normal Mouse Tissue Array2A7.C11-FITC Hu-IgG1-FITC Tissue Types 2 μg/ml 5 μg/ml 5 μg/ml Skin, earlobe Epidermis − ± − Sabaceous gland − ± − Other elements − − − ColonSurface epithelium ±, 1+ 1+ ± Other elements − − − Small Intestine Cryptepithelium ±, 1+ 1+, 2+ ± Other elements − − − Stomach Surface &glandular 1+, 2+, ocas 1+, 2+, freq 1+, 2+, ocas epithelial cells Nerveplexus − ±, 1+ − Other elements − − − Pancreas Acinar epithelium 1+ 2+±, 1+ Islets − ± − Other elements − − − Salivary gland Acinar epithelium± 1+ − Other elements − − − Liver Hepatocytes − ±, − − Other elements −− − Cerebrum Neurons ± 2+, 1+, freq ±, − Neuropil/fibers −, ± 2+, 1+,ocas − Pons Neurons ± ± ± Neuropil/fibers ± 2+, 1+, freq − CerebelleumPurkinje cells ±, 1+ 1+ ±, − White matter − 1+, 2+ − Other elements − −− Spleen Large lymphoid − 1+, 2+, rare − cells in red pulp Otherelements − −, ± − Thymus − − − Skeletal muscle − − − Tongue − − − Heart− −, ± − Lung − − − Kidney cortex − −, ± − Kidney medulla − − − Urinarybladder Transitional − ±, 1+ − epithelium Other elements − − − Seminalvesicle Epithelium ±, − ± − Fluid in the lumen 1+ 3+ ± Other elements −− − Testis Primary − ±, 1+ − Spermotocytes Other elements − − −Epididymis − − − Uterus Endometrium/gland −, ± ± − epithelium Otherelements − − − Ovary − ± − Intensity of immunoreactivity: +−(equivocal); + (weak); 2+ (moderate); 3+ (strong); 4+ (intense); −(negative). Freq: frequent; Ocas: occasional

These data and corresponding data collected for anti-O8E antibodies 1G11and 2F9, demonstrate that strong to intense O8E immunoreactivity (3+,4+) was present in enteroendocrine-like cells in colon and smallintestine, as well as in the lumen fluid of seminary vesicle; weak tomoderate O8E immunoreactivity (1+, 2+) was revealed in neurons ofcerebrum, in neuropils and fibers of cerebrum and pons, in the whitematter of cerebellum, in the crypt epithelial cells of small intestineand in a small number of large lymphoid cells in the spleen; weak O8Eimmunoreactivity (1+) was demonstrated in colon surface epithelium,Purkinje cells in cerebellum and acinar epithelium of salivary gland andpancreas; equivocal to weak O8E immunoreactivity was shown intransitional epithelium of urinary bladder, primary spermotocytes oftestis and nerve plexus in stomach; and all other organs exhibitnegative to equivocal staining, which include skin, liver, heart, lung,thymus, kidney, uterus, ovary, epididymis, tongue and skeletal muscles.

Example 11 Production of Defucosylated HuMAbs

This Example demonstrates the production of anti-O8E HuMAbs lacking infucosyl residues.

Antibodies with reduced amounts of fucosyl residues have beendemonstrated to increase the ADCC ability of the antibody. The CHO cellline Ms704-PF, which lacks the fucosyltransferase gene FUT 8 (Biowa,Inc., Princeton, N.J.), is electroporated with a vector that expressesthe heavy and light chains of an anti-O8E HuMAb. Drug-resistant clonesare selected by growth in Ex-Cell 325-PF CHO media (JRH Biosciences,Lenexa, Kans.) with 6 mM L-glutamine and 500 μg/ml G418 (Invitrogen,Carlsbad, Calif.). Clones are screened for IgG expression by standardELISA assay. Two separate clones are produced, B8A6 and B8C1, which hasproduction rates ranging from 1.0 to 3.8 picograms per cell per day.

Example 12 Assessment of ADCC Activity of Defucosylated Anti-O8EAntibody

This Example discloses the testing of defucosylated andnon-defucosylated anti-O8E monoclonal antibodies for the ability to killO8E⁺ cells in the presence of effector cells via antibody dependentcellular cytotoxicity (ADCC) in a fluorescence cytotoxicity assay.

Human anti-O8E monoclonal antibodies are defucosylated as describedabove. Human effector cells are prepared from whole blood as follows.Human peripheral blood mononuclear cells are purified from heparinizedwhole blood by standard Ficoll-paque separation. The cells areresuspended in RPMI1640 media containing 10% FBS (culture media) and 200U/ml of human IL-2 and incubated overnight at 37° C. The following day,the cells are collected and washed once in culture media and resuspendedat 2×10⁷ cells/ml. Target O8E+ cells are incubated with BATDA reagent(Perkin Elmer, Wellesley, Mass.) at 2.5 μl BATDA per 1×10⁶ targetcells/mL in culture media supplemented with 2.5 mM probenecid (assaymedia) for 20 minutes at 37° C. The target cells are washed four timesin PBS with 20 mM HEPES and 2.5 mM probenecid, spun down and brought toa final volume of 1×10⁵ cells/ml in assay media.

The O8E+ cell line ARH-77 (human B lymphoblast leukemia; ATCC AccessionNo. CRL-1621) is tested for antibody specific ADCC to the defucosylatedand non-defucosylated human anti-O8E monoclonal antibody using theDelfia fluorescence emission analysis as follows. The target cell lineARH77 (100 μl of labeled target cells) is incubated with 50 μl ofeffector cells and 50 μl of either 1G11 or defucosylated 1G11 antibody.A target to effector ratio of 1:100 is used throughout. A human IgG1isotype control is used as a negative control. Following a 2100 rpmpulse spin and one hour incubation at 37° C., the supernatants arecollected, quick spun again and 20 μl of supernatant is transferred to aflat bottom plate, to which 180 μl of Eu solution (Perkin Elmer,Wellesley, Mass.) is added and read in a Fusion Alpha TRF plate reader(Perkin Elmer). The % lysis is calculated as follows: (samplerelease−spontaneous release*100)/(maximum release−spontaneous release),where the spontaneous release is the fluorescence from wells which onlycontain target cells and maximum release is the fluorescence from wellscontaining target cells and have been treated with 3% Lysol. TheO8E+expressing cell line ARH-77 will show an antibody mediatedcytotoxicity with the HuMAb anti-O8E antibody 1G11 and an increasedpercentage of specific lysis associated with the defucosylated form ofthe anti-O8E antibody G101. Thus, defucosylated HuMAb anti-O8Eantibodies increase specific cytotoxicity to O8E+ expressing cells.

Example 13 Internalization of HuMab Anti-O8E Antibodies byImmuno-Fluorescence Staining Analysis

The target cell lines, O8E⁺ SKBR3 (human breast cancer, ATCC#1 HTB-30)and ZR-75 (human breast cancer, ATCC# CRL-1500) were used to test forinternalization of HuMab anti-08E antibodies 2A7C11, 1G11H1 and 2F9E6upon binding to the cells using immuno-fluorescence staining.

SKBR3 and ZR-75 cells (10⁴ per 100 μl per well in 96-well plate),harvested from tissue culture flask by treatment with 0.25%Trypsin/EDTA, were incubated with each of HuMab anti-O8E antibodies at 5μg/ml in FACS buffer (PBS+5% FBS, media) for 30 minutes on ice. A humanIgG1 isotype control was used as a negative control. Following 2× washeswith the media, the cells were re-suspended in the media (100 μl perwell) and then incubated with goat anti-human secondary antibodyconjugated with PE (Jackson ImmunoResearch Lab) at 1:00 dilution on icefor 30 minutes. Following washed with the media, the cells were eitherimmediately imaged under a fluorescent microscope (Nikon) at 0 min orincubated at 37° C. for various times. The images of cell morphology andimmuno-fluorescence intensity of the stained cells were taken atdifferent time points as indicated in the figures below. Thefluorescence was only observed in the cells stained with HuMab anti-O8Eantibodies. No fluorescence was detected with the IgG1 control antibody.Similar results were also obtained with FITC-direct conjugated HuMabanti-O8E antibodies in the assays.

The imaging data showed the appearance of the fluorescence on cellsurface membrane with all three HuMab anti-O8E antibodies at 0 min. In30 min incubation, the membrane fluorescence intensity significantlydecreased while staining increased inside of the cells. At the 120 minpoint, the fluorescence on the membrane disappeared and instead appearedto be present in intracellular compartments. The data demonstrates thatHuMab anti-O8E antibodies can be specifically internalized upon bindingto O8E-expressing endogenous tumor cells.

Example 14 Efficacy of Anti-O8E Antibodies on HEK-B7H4 Tumors in SCIDMice

In this Example, SCID mice implanted with HEK-B7H4 tumors are treated invivo with naked anti-O8E antibodies to examine the in vivo effect of theantibodies on tumor growth.

Severe combined immune deficient (SCID) mice, which lack functional 13and T lymphocytes were used to study tumor growth. Cells from the HEKtumor cell line transfected with B7H4 were implanted subcutaneously at 5million cells/mouse in matrigel (50% v/v). Each mouse received aninoculum of 0.2 ml of cells on day 0. The mice were checked for tumorgrowth starting at day 10 and monitored twice weekly for tumor growthfor approximately 6 weeks. When tumors reached about 130 mm³, the micewere randomized by tumor volume into 3 groups. The mice were treatedeither with 10 mg/kg naked anti-O8E antibody 2A7, an isotype controlantibody or formulation buffer as a negative control. The animals weredosed by intraperitoneal injection every 5 days for 5 injections. Usingan electronic caliper, the tumors were measured three dimensionally(height×width×length) and tumor volume was calculated. Mice wereeuthanized when tumors reached a volume of 1500 mm³ or showed greaterthan 15% weight loss. The results are shown in FIG. 20. Tumor growth wasinhibited by treatment with the anti-O8E antibody 2A7. The median tumorgrowth inhibition for the group treated with 2A7 was 63% on day 34. Thetumors resumed growth after the dosing was stopped. These results showthat anti-O8E antibodies are effective in treating tumors that expressO8E in vivo.

Example 15 Immunohistochemistry Using an Anti-O8E Antibody

The ability of the anti-B7H4 HuMAb 2A7 to recognize B7H4 byimmunohistochemistry was examined using clinical biopsies from ovariancancer, lung cancer, breast cancer, and head & neck cancer

For immunohistochemistry, 5 μm frozen sections were used (Ardais Inc,USA). After drying for 30 minutes, sections were fixed with acetone (atroom temperature for 10 minutes) and air-dried for 5 minutes. Slideswere rinsed in PBS and then pre-incubated with 10% normal goat serum inPBS for 20 min and subsequently incubated with 10 μg/ml fitcylatedantibody in PBS with 10% normal goat serum for 30 min at roomtemperature. Next, slides were washed three times with PBS and incubatedfor 30 min with mouse anti-FITC (10 μg/ml DAKO) at room temperature.Slides were washed again with PBS and incubated with Goat anti-mouse HRPconjugate (DAKO) for 30 minutes at room temperature. Slides were washedagain 3× with PBS. Diaminobenzidine (Sigma) was used as substrate,resulting in brown staining. After washing with distilled water, slideswere counter-stained with hematoxyllin for 1 min. Subsequently, slideswere washed for 10 secs in running distilled water and mounted inglycergel (DAKO). Clinical biopsy immunohistochemical staining displayedpositive staining in the lung cancer, breast cancer, ovarian cancer, andhead & neck cancer samples.

Example 16 Quantitative RT-PCR on Normal and Cancer Tissues

Various normal and cancerous tissue samples were screened for O8E mRNAexpression using quantitative reverse transcriptase PCR (RT-PCR).Expression of mRNA is indicative of O8E protein expression.

For quantitative RT-PCR, the following O8E primers were used: B7-H4.3:AGGATGGAATCCTGAGCTGCACTT; B7-H4.4: TCCGACAGCTCATCTTTGCCTTCT as providedby Operon (Huntsville, Ala.). Standard reaction conditions were used (5μl cDNA template at 1 ng/μl, 0.1 μl upstream primer at 40 μM, 0.1 μldownstream primer at 40CM, 6 μl 2×SYBR Green PCR mix (Applied Biosystems# 4367659), and 0.8 μl water). The cDNA was amplified for 40 cyclesusing standard PCR conditions in an ABI Prism 7900HT (AppliedBiosystems, Foster City, Calif.). The quantitative RT-PCR results areshown in Table 7 below. Samples with undetermined counts representvalues that were below a fluorescence threshold. Breast, ovarian andhead and neck tumors were shown to express O8E, with the highest levelsof expression seen in some ovarian and head and neck cancer samples.This demonstrates that there is increased expression of O8E in breast,ovarian and head and neck tumor samples relative to normal tissue.

TABLE 7 Quantitative RT-PCR expression in normal and cancer tissuesTissue Count Quantity N.Adipose (#301) 28.953062 25.57793 N.Artery(#303) 31.856901 3.0423617 N.Bladder (#257) 30.620392 7.5326214 N.BoneMarrow (#342) Undetermined 0 N.Brain (#258) 34.33955 0.49280354 N.Breast(#259) 25.63064 292.28528 N.Colon (#261) Undetermined 0 N.Esophagus(#262) 32.27514 2.2388945 N.Heart (#125) Undetermined 0 N.Kidney (#264)33.599422 0.8479082 N.Liver (#266) Undetermined 0 N.Lung (#268) 32.445231.9763907 N.Lymph Node (#315) Undetermined 0 N.Ovary (#270) 35.0457040.29364112 N.Pancreas (#271) 28.446985 37.06916 N.Peripheral BloodLeukocytes (#302) 34.652363 0.39180183 N.Prostate (#272) 32.6359941.7184163 N.Retina (#256) 34.70426 0.37717298 N.Skeletal Muscle (#119)Undetermined 0 N.Skeletal Muscle (#126) Undetermined 0 N.Skin (#273)Undetermined 0 N.Spinal Cord (#129) 39.383526 0.01220525 N.Spleen (#274)Undetermined 0 N.Stomach (#275) Undetermined 0 N.Tongue (#324) 30.9567585.886249 N.Tonsil (#325) Undetermined 0 N.Trachea (#314) 29.77134314.03797 Breast T. (#176) 33.798374 0.7328206 Breast T. (#177) 25.759022266.02777 Breast T. (#178) 28.572468 33.81085 Breast T. (#179) 25.31508368.374 Breast T. (#180) 29.323488 19.494516 Head/Neck T. (Larynx, #402)28.116425 47.23582 Head/Neck T. (Pharynx, #403) 25.776083 262.72076Head/Neck T. (Tongue, #403) 26.950275 111.07142 Head/Neck T. (Tonsil,#404) 23.03704 1957.3722 Kidney T. (#167) 27.029814 104.77927 Ovary T.(#187) 25.321087 366.75525 Ovary T. (#188) 22.846964 2250.0833 Ovary T.(#189) 25.079527 437.81958 Ovary T. (#190) 27.964441 52.80399 Ovary T.(#191) 22.686525 2530.9656

The present disclosure is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of thisdisclosure in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Patents, patent applications, publications, product descriptions andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

1. An isolated human monoclonal antibody or an antigen-binding portionthereof, wherein the antibody: (a) binds to human O8E with a K_(D) of1×10⁻⁷ M or less; and (b) binds to a breast cell carcinoma tumor cellline.
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. The antibody of claim 1, wherein said antibody isinternalized.
 8. The antibody of claim 1, wherein the breast cellcarcinoma tumor cell line is selected from the group consisting of theSKBR3 cell line.
 9. The antibody of claim 1, wherein said antibody lacksfucose residues.
 10. An isolated monoclonal antibody or antigen bindingportion thereof, wherein the antibody cross-competes for binding to O8Ewith a reference antibody, wherein the reference antibody: (a) binds tohuman O8E with a K_(D) of 1×10⁻⁷ M or less; and (b) binds to a breastcell carcinoma tumor cell line.
 11. The antibody of claim 10, whereinthe reference antibody comprises: (a) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 1; and (b) a lightchain variable region comprising the amino acid sequence of SEQ ID NO:6.
 12. The antibody of claim 10, wherein the reference antibodycomprises: (a) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 2; and (b) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:
 7. 13. The antibody ofclaim 10, wherein the reference antibody comprises: (a) a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 3; and(b) a light chain variable region comprising the amino acid sequence ofSEQ ID NO:
 8. 14. The antibody of claim 10, wherein the referenceantibody comprises: (a) a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 4; and (b) a light chain variableregion comprising the amino acid sequence of SEQ ID NO:
 9. 15. Theantibody of claim 10, wherein the reference antibody comprises: (a) aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 5; and (b) a light chain variable region comprising the amino acidsequence of SEQ ID NO:
 10. 16. An isolated monoclonal antibody or anantigen-binding portion thereof, comprising a heavy chain variableregion that is the product of or derived from a human V_(H) 4-34 gene, ahuman V_(H) 3-53 gene or a V_(H) 3-9 gene, and a light chain variableregion that is the product of or derived from a human V_(K) A17 gene orhuman V_(K) L6 gene, wherein the antibody specifically binds O8E. 17.(canceled)
 18. The antibody of claim 1, which comprises: a) a heavychain variable region CDR1 comprising SEQ ID NO: 11; b) a heavy chainvariable region CDR2 comprising SEQ ID NO: 16; c) a heavy chain variableregion CDR3 comprising SEQ ID NO: 21; d) a light chain variable regionCDR1 comprising SEQ ID NO: 26; e) a light chain variable region CDR2comprising SEQ ID NO: 31; and f) a light chain variable region CDR3comprising SEQ ID NO:
 36. 19. The antibody of claim 1, which comprises:a) a heavy chain variable region CDR1 comprising SEQ ID NO: 12; b) aheavy chain variable region CDR2 comprising SEQ ID NO: 17; c) a heavychain variable region CDR3 comprising SEQ ID NO: 22; d) a light chainvariable region CDR1 comprising SEQ ID NO: 27; e) a light chain variableregion CDR2 comprising SEQ ID NO: 32; and f) a light chain variableregion CDR3 comprising SEQ ID NO:
 37. 20. The antibody of claim 1, whichcomprises: a) a heavy chain variable region CDR1 comprising SEQ ID NO:13; b) a heavy chain variable region CDR2 comprising SEQ ID NO: 18; c) aheavy chain variable region CDR3 comprising SEQ ID NO: 23; d) a lightchain variable region CDR1 comprising SEQ ID NO: 28; e) a light chainvariable region CDR2 comprising SEQ ID NO: 33; and f) a light chainvariable region CDR3 comprising SEQ ID NO:
 38. 21. The antibody of claim1, which comprises: a) a heavy chain variable region CDR1 comprising SEQID NO: 14; b) a heavy chain variable region CDR2 comprising SEQ ID NO:19; c) a heavy chain variable region CDR3 comprising SEQ ID NO: 24; d) alight chain variable region CDR1 comprising SEQ ID NO: 29; e) a lightchain variable region CDR2 comprising SEQ ID NO: 34; and f) a lightchain variable region CDR3 comprising SEQ ID NO:
 39. 22. The antibody ofclaim 1, which comprises: a) a heavy chain variable region CDR1comprising SEQ ID NO: 15; b) a heavy chain variable region CDR2comprising SEQ ID NO: 20; c) a heavy chain variable region CDR3comprising SEQ ID NO: 25; d) a light chain variable region CDR1comprising SEQ ID NO: 30; e) a light chain variable region CDR2comprising SEQ ID NO: 35; and f) a light chain variable region CDR3comprising SEQ ID NO:
 40. 23. The isolated monoclonal antibody orantigen binding portion thereof of claim 1, comprising: a) a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 1; andb) a light chain variable region comprising the amino acid sequence ofSEQ ID NO:
 6. 24. The isolated monoclonal antibody or antigen bindingportion thereof of claim 1, comprising: a) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 2; and b) a light chainvariable region comprising the amino acid sequence of SEQ ID NO:
 7. 25.The isolated monoclonal antibody or antigen binding portion thereof ofclaim 1, comprising: a) a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 3; and b) a light chain variableregion comprising the amino acid sequence of SEQ ID NO:
 8. 26. Theisolated monoclonal antibody or antigen binding portion thereof of claim1, comprising: a) a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 4; and b) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:
 9. 27. The isolatedmonoclonal antibody or antigen binding portion thereof of claim 1,comprising: a) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 5; and b) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:
 10. 28. (canceled) 29.An immunoconjugate comprising the antibody or antigen-binding portionthereof, of claim 1, linked to a therapeutic agent.
 30. A compositioncomprising the immunoconjugate of claim 29 and a pharmaceuticallyacceptable carrier.
 31. The immunoconjugate of claim 30, wherein thetherapeutic agent is a cytotoxin.
 32. (canceled)
 33. The immunoconjugateof claim 29, wherein the therapeutic agent is a radioactive isotope. 34.(canceled)
 35. An isolated nucleic acid molecule encoding the antibodyor antigen-binding portion thereof, of claim
 1. 36. An expression vectorcomprising the nucleic acid molecule of claim
 35. 37. A host cellcomprising the expression vector of claim
 36. 38. A method for preparingan anti-O8E antibody which comprises expressing the antibody in the hostcell of claim 39 and isolating the antibody from the host cell.
 39. Amethod of treating or preventing a disease characterized by growth oftumor cells expressing O8E, comprising administering to a subject theantibody or antigen-binding portion thereof, of claim 1 in an amounteffective to treat or prevent the disease.
 40. The method of claim 39,wherein the disease is cancer.
 41. The method of claim 40, wherein thecancer is selected from the group consisting of breast cell carcinoma,ovarian cancer, kidney cancer, and head and neck cancer. 42-44.(canceled)